Microfluidic systems including three-dimensionally arrayed channel networks

ABSTRACT

The present invention provides, in certain embodiments, improved microfluidic systems and methods for fabricating improved microfluidic systems, which contain one or more levels of microfluidic channels. The inventive methods can provide a convenient route to topologically complex and improved microfluidic systems. The microfluidic systems provided according to the invention can include three-dimensionally arrayed networks of fluid flow paths therein including channels that cross over or under other channels of the network without physical intersection at the points of cross over. The microfluidic networks of the invention can be fabricated via replica molding processes, also provided by the invention, utilizing mold masters including surfaces having topological features formed by photolithography. The microfluidic networks of the invention are, in some cases, comprised of a single replica molded layer, and, in other cases, are comprised of two, three, or more replica molded layers that have been assembled to form the overall microfluidic network structure. The present invention also describes various novel applications for using the microfluidic network structures provided by the invention.

RELATED APPLICATIONS

This application is a continuation of PCT International Application No.PCT/US01/16973 filed May 25, 2001, which was published under PCT Article21(2) in English and is a continuation-in-part of U.S. application Ser.No. 09/578,589, filed May 25, 2000. Both applications are herebyincorporated by reference.

FIELD OF INVENTION

The present invention involves microfluidic network structures, methodsfor fabricating microfluidic network structures, and methods for usingsuch structures.

BACKGROUND OF THE INVENTION

The need for complexity in microfluidic systems is increasing rapidly assophisticated functions—chemical reactions and analyses, bioassays,high-throughput screens, and sensors—are being integrated into singlemicrofluidic devices. Complex systems of channels require more complexconnectivity than can be generated in conventional two-dimensionalmicrofluidic systems having a single level of channels, since suchtypical single-level designs do not allow two channels to cross withoutfluidically connecting. Most methods for fabricating microfluidicchannels are based on photolithographic procedures, and yield suchtwo-dimensional systems. There are a number of more specializedprocedures, such as stereolithography (see for example, K. Ikuta, K.Hirowatari, T. Ogata, Proc. IEEE MEMS '94, Oiso, Japan, Jan. 25-28,1994, pp. 1-6), laser-chemical three-dimensional writing (see forexample, T. M. Bloomstein, D. J. Ehrlich, J. Vac. Sci. Technol. B, Vol.10, pp. 2671-2674, 1992), and modular assembly (see for example, C.Gonzalez, R. L. Smith, D. G. Howitt, S. D. Collins, Sens. Actuators A,Vol. 66, pp. 315-332, 1998), that yield three-dimensional structures,but these methods are typically time consuming, difficult to perform,and expensive, and are thus not well suited for either prototyping ormanufacturing, and are also not capable of making certain types ofstructures. Better methods for generating complex three-dimensionalmicrofluidic systems are needed to accelerate the development ofmicrofluidic technology. The present invention, in some embodiments,provides such improved methods for generating complex three-dimensionalmicrofluidic systems.

It is known to use a stamp or mold to transfer patterns to a surface ofa substrate (see for example, R. S. Kane, S. Takayama, E. Ostuni, D. E.Ingber, G. M. Whitesides, Biomaterials, Vol. 20, pp. 2363-2376, 1999;and Y. Xia, G. M. Whitesides, Angew. Chem. Int. Ed. Engl., Vol. 37, pp.551-575, 1998; U.S. Pat. No. 5,512,131; International Pat. PublicationNo. WO 97/33737, published Sep. 18, 1997). Most conventional softlithographic techniques, for example, microcontact printing (μCP) (seefor example, C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, D. E.Ingber, Science, Vol. 276, pp. 1425-1428, 1997; A. Bernard, E.Delamarche, H. Schmid, B. Michel, H. R. Bosshard, H. Biebuyck, Langmuir,Vol. 14, pp. 2225-2229, 1998) and micromolding in capillaries (MIMIC)(see for example, N. L. Jeon, I. S. Choi, B. Xu, G. M. Whitesides, Adv.Mat., Vol. 11, pp. 946-949, 1999; E. Delamarche, A. Bernard, H. Schmid,B Michel, H. Biebuyck, Science, Vol. 276, pp. 779-781, 1997; E.Delamarche, A. Bernard, H. Schmid, A. Bietsch, B. Michel, h. Biebuyck,J. Am. Chem. Soc., Vol. 120, pp. 500-508, 1998; A. Folch, A. Ayon, O.Hurtado, M. A. Schmidt, M. Toner, J. Biomech. Eng., Vol. 121, pp. 28-34,1999; A. Folch, M. Toner, Biotech. Prog., Vol. 14, pp. 388-392, 1998),have been limited to procedures that pattern one substance at a time, orto relatively simple, continuous patterns. These constraints are bothtopological and practical. The surface of a stamp in μCP, or of achannel system in MIMIC, is effectively a two-dimensional structure. InμCP, this two-dimensionality of the stamp limits the types of patternsthat can be transferred to those comprising a single “color” of ink inthe absence of a way of selectively “inking” different regions of thestamp with different materials. Patterning of multiple “inks” usingconventional methods requires multiple steps of registration andstamping. In MIMIC, the two-dimensional channel system limits patterningto relatively simple, continuous structures or requires multiplepatterning steps.

There remains a general need in the art for improved methods for formingpatterns on surfaces with soft lithographic techniques, and forproviding techniques able to pattern onto a surface arbitrarytwo-dimensional patterns and able to form complex patterns comprised ofmultiple regions, where different regions of the pattern can comprisedifferent materials, on a surface without the need for multiple steps ofregistration or stamping and without the need to selectively “ink”different regions of the stamp with different materials. The presentinvention, in some embodiments, provides such improved methods forforming patterns on surfaces with soft lithographic techniques.

SUMMARY OF THE INVENTION

The present invention involves, in certain embodiments, improvedmicrofluidic systems and procedures for fabricating improvedmicrofluidic systems, which contain one or more levels of microfluidicchannels. The inventive methods can provide a convenient route totopologically complex and improved microfluidic systems. The presentinvention also, in some embodiments, involves microfluidic systems andmethods for fabricating complex patterns of materials, such asbiological materials and cells, on surfaces. In such embodiments, theinvention involves microfluidic surface patterning systems and methodsfor fabricating complex, discontinuous patterns on surfaces that canincorporate or deposit multiple materials onto a surface. The presentinvention, in some embodiments, can provide improved stamps formicrocontact surface patterning able to pattern onto a surface arbitrarytwo-dimensional patterns and able to pattern multiple substances onto asurface without the need for multiple steps of registration or stampingduring patterning and without the need to selectively “ink” differentregions of the stamp with different materials.

According to one embodiment of the invention, a microfluidic network isdisclosed. The microfluidic network comprises a polymeric structureincluding therein at least a first and a second non-fluidicallyinterconnected fluid flow paths. At least the first flow path comprisesa series of interconnected channels within the polymeric structure. Theseries of interconnected channels includes at least one first channeldisposed within a first level of the structure, at least one secondchannel disposed within a second level of the structure, and at leastone connecting channel fluidically interconnecting the first channel andthe second channel. At least one channel within the structure has across-sectional dimension not exceeding about 500 μm. The structureincludes at least one channel disposed within the first level of thestructure that is non-parallel to at least one channel disposed withinthe second level of the structure.

In another embodiment of the invention, a microfluidic network isdisclosed. The microfluidic network comprises an elastomeric structureincluding therein at least one fluid flow path. The flow path comprisesa series of interconnected channels within the structure. The series ofinterconnected channels includes at least one first channel disposedwithin a first level of the structure, at least one second channeldisposed within a second level of the structure, and at least oneconnecting channel fluidically interconnecting the first channel and thesecond channel. At least one channel within the structure has across-sectional dimension not exceeding about 500 μm, and the structureincludes at least one channel disposed within the first level of thestructure that is non-parallel to at least one channel disposed withinthe second level of the structure.

In yet another embodiment, a polymeric membrane is disclosed. Thepolymeric membrane comprises a first surface including at least onechannel disposed therein, a second surface including at least onechannel disposed therein, and a polymeric region intermediate the firstsurface and the second surface. The intermediate region includes atleast one connecting channel therethrough fluidically interconnectingthe channel disposed in the first surface and the channel disposed inthe second surface of the membrane. At least one channel has across-sectional dimension not exceeding about 500 μm.

In another embodiment of the invention, a method for forming amicrofluidic network structure is disclosed. The method comprisesproviding at least one mold substrate, forming at least one topologicalfeature on a surface of the mold substrate to form a first mold master,contacting the surface with a first hardenable liquid, hardening theliquid thereby creating a first molded replica of the surface, removingthe first molded replica from the first mold master, and assembling thefirst molded replica into a structure comprising a microfluidic network.The assembled microfluidic network structure has at least one fluid flowpath comprising a series of interconnected channels within thestructure. The series of interconnected channels includes at least onefirst channel disposed within a first level of the structure, at leastone second channel disposed within a second level of the structure, andat least one connecting channel fluidically interconnecting the firstchannel and the second channel. At least one of the channels within thestructure has a cross-sectional dimension not exceeding about 500 μm.The structure includes at least one channel disposed within the firstlevel of the structure that is non-parallel to at least one channeldisposed within the second level of the structure.

In yet another embodiment, a method for forming a molded structure isdisclosed. The method comprises providing at least one mold substrateand forming at least one two-level topological feature having at leastone lateral dimension not exceeding 500 μm on a surface of the substrateto form a mold master. The two-level topological feature ischaracterized by a first portion having a first depth or height withrespect to a region of the surface adjacent to the feature, and a secondportion integrally connected with the first portion having a seconddepth or height with respect to the region of the surface adjacent tothe feature that is greater than the first depth or height. The methodfurther comprises contacting the surface with a hardenable liquid,hardening the liquid thereby creating a molded replica of the surface,and removing the molded replica from the mold master.

In another embodiment of the invention, a method for forming topologicalfeatures on a surface of a material is disclosed. The method comprisesexposing portions of a surface of a first layer of photoresist toradiation in a first pattern, coating the surface of the first layer ofphotoresist with a second layer of photoresist, exposing portions of asurface of the second layer of photoresist to radiation in a secondpattern different from the first pattern, and developing the first andsecond photoresist layers with a developing agent. The developing stepyields a positive relief pattern in photoresist that includes at leastone two-level topological feature having at least one cross-sectionaldimension not exceeding 500 μm. The two-level topological feature ischaracterized by a first portion having a first height with respect tothe surface of the material and a second portion, integrally connectedto the first portion, having a second height with respect to the surfaceof the material.

In yet another embodiment, a method for forming a molded structure isdisclosed. The method involves providing a first mold master having asurface formed of an elastomeric material and including at least onetopological feature with at least one cross-sectional dimension notexceeding about 500 μm thereon. The method further comprises providing asecond mold master having a surface including at least one topologicalfeature with at least one cross-sectional dimension not exceeding about500 μm thereon. The method further comprises placing a hardenable liquidin contact with the surface of at least one of the first and second moldmaster, bringing the surface of the first mold master into at leastpartial contact with the surface of the second mold master, hardeningthe liquid thereby creating a molded replica of the surface of the firstmold master and the surface of the second mold master, and removing themolded replica from at least one of the mold masters.

In another embodiment of the invention, a method for forming a moldedstructure is disclosed. The method involves providing a first moldmaster having a surface including at least a first topological featurewith at least one cross-sectional dimension not exceeding about 500 μmthereon and at least a second topological feature comprising a firstalignment element. The method further comprises providing a second moldmaster having a surface including at least a first topological featurewith at least one cross-sectional dimension not exceeding about 500 μmthereon and at least a second topological feature comprising a secondalignment element having a shape that is mateable to the shape of thefirst alignment element. The method further comprises placing ahardenable liquid in contact with the surface of at least one of thefirst and second mold master, bringing the surface of the first moldmaster into at least partial contact with the surface of the second moldmaster, aligning the first topological features of the first and secondmold masters with respect to each other by adjusting a position of thefirst mold master with respect to a position of the second mold masteruntil the first alignment element matingly engages and interdigitateswith the second alignment element, hardening the liquid thereby creatinga molded replica of the surface of the first mold master and the surfaceof the second mold master, and removing the molded replica from at leastone of the mold masters.

In yet another embodiment of the invention, a method for aligning andsealing together surfaces is disclosed. The method comprises disposingtwo surfaces, at least one of which is oxidized, adjacent to each othersuch that they are separated from each other by a continuous layer of aliquid that is essentially non-reactive with the surfaces, aligning thesurfaces with respect to each other, and removing the liquid frombetween the surfaces, thereby sealing the surfaces together via achemical reaction between the surfaces.

In another embodiment of the invention, a method for molding an articleis disclosed. The method comprises providing a first mold master havinga surface with a first set of surface properties and providing a secondmold master having a surface with a second set of surface properties. Atleast one of the first and second mold masters has a surface includingat least one topological feature with at least one cross-sectionaldimension not exceeding about 500 μm thereon. The method furthercomprises placing a hardenable liquid in contact with the surface of atleast one of the first and second mold masters, bringing the surface ofthe first mold master into at least partial contact with the surface ofthe second mold master, hardening the liquid thereby creating a moldedreplica of the surface of the first mold master and the surface of thesecond mold master, separating the mold masters from each other, andremoving the molded replica from the surface of the first mold masterwhile leaving the molded replica in contact with and supported by thesurface of the second mold master.

In yet another embodiment, a microfluidic network is disclosed. Themicrofluidic network comprises a polymeric structure including thereinat least a first and a second non-fluidically interconnected fluid flowpaths. The first flow path comprises at least two non-colinearinterconnected channels disposed within a first plane, and the secondflow path comprises at least one channel disposed within a second planethat is non-parallel with the first plane. At least one channel withinthe structure has a cross-sectional dimension not exceeding about 500μm.

In another embodiment of the invention, a microfluidic network isdisclosed. The microfluidic network comprises a polymeric structureincluding therein at least one fluid flow path. The fluid flow path isformed of at least one channel and has a longitudinal axis defined bythe direction of bulk fluid flow within the flow path. The longitudinalaxis of the flow path is not disposed within any single plane.

In another embodiment of the invention, a method of patterning amaterial surface is disclosed. The method comprises providing a stamphaving a structure including at least one flow path comprising a seriesof interconnected channels within the structure. The series ofinterconnected channels includes at least one first channel disposedwithin an interior region of the structure, at least one second channeldisposed within a stamping surface of the structure defining a firstpattern therein, and at least one connecting channel fluidicallyinterconnecting the first channel and the second channel. The methodfurther comprises contacting the stamping surface with a portion of thematerial surface, and, while maintaining the stamping surface in contactwith the portion of the material surface, at least partially filling theflow path with a fluid so that at least a portion of the fluid contactsthe material surface.

In yet another embodiment, a method of patterning a material surface isdisclosed. The method comprises providing a stamp having a structureincluding at least two non-fluidically interconnected flow paths thereinincluding a first fluid flow path defining a first pattern of channelsdisposed within a stamping surface of the structure and a second fluidflow path defining a second pattern of channels disposed within thestamping surface of the structure. Each of the first and second patternsof channels is non-continuous, and the channels defining the firstpattern are non-intersecting with the channels defining the secondpattern. The method further comprises contacting the stamping surfacewith a portion of the material surface, while maintaining the stampingsurface in contact with the portion of the material surface, at leastpartially filling the first flow path with a first fluid so that atleast a portion of the first fluid contacts the material surface and atleast partially filling the second flow path with a second fluid so thatat least a portion of the second fluid contacts the material surface,and removing the stamping surface to provide a pattern on the materialsurface according to the first pattern, which is formed by contact ofthe material surface with the first fluid, and according to the secondpattern, which is formed by contact of the material surface with thesecond fluid.

In another embodiment, a method of patterning a material surface isdisclosed, the method involves providing a stamp having a structureincluding at least one non-linear fluid flow path therein in fluidcommunication with a stamping surface of the structure. The methodfurther involves contacting the stamping surface with a portion of thematerial surface and, while maintaining the stamping surface in contactwith the portion of the material surface, at least partially filling theflow path with a fluid so that at least a portion of the fluid contactsthe material surface.

Other advantages, novel features, and objects of the invention willbecome apparent from the following detailed description of the inventionwhen considered in conjunction with the accompanying drawings, which areschematic and which are not intended to be drawn to scale. In thefigures, each identical or nearly identical component that isillustrated in various figures is represented by a single numeral. Forpurposes of clarity, not every component is labeled in every figure, noris every component of each embodiment of the invention shown whereillustration is not necessary to allow those of ordinary skill in theart to understand the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a perspective view of a schematic illustration of amicrofluidic network structure having a series of interconnectedchannels arranged in a “basketweave” configuration;

FIG. 1 b is a two-dimensional projection of the microfluidic networkstructure of FIG. 1 a;

FIG. 2 a is a perspective view of a schematic illustration of a secondembodiment of a microfluidic network structure;

FIG. 2 b is a two-dimensional projection of the microfluidic networkstructure of FIG. 2 a;

FIG. 3 a is a perspective view of a schematic illustration of a thirdembodiment of a microfluidic network structure;

FIG. 3 b is a two-dimensional projection of the microfluidic networkstructure of FIG. 3 a;

FIG. 4 a is a perspective view of a schematic illustration of afive-level microfluidic network comprising a centrally disposed straightchannel surrounded by a coiled fluid flow path;

FIG. 4 b is a two-dimensional projection of the microfluidic networkstructure of FIG. 4 a;

FIGS. 5 a-5 c are schematic illustrations of one embodiment of thefabrication method for forming a microfluidic network structureaccording to one embodiment of the invention;

FIGS. 6 a-6 c are schematic illustrations of one embodiment of aself-aligning method provided by the invention;

FIG. 6 d is a schematic illustration of a replica molded layer of amicrofluidic network having a perimetric shape for use in one embodimentof a self-aligning method according to the invention;

FIG. 7 is a schematic illustration of a second embodiment of amicrofluidic network fabrication method according to the invention;

FIG. 8 is a schematic illustration of a method for forming a two-leveltopological feature on a surface of the substrate by photolithographyprovided according to the invention;

FIGS. 9 a-9 b are schematic illustrations of a third embodiment forforming a microfluidic network structure according to the invention;

FIG. 9 c is a series of schematic, cross-sectional illustrations of amodification of the third embodiment for forming the microfluidicnetwork structure of FIGS. 9 a-9 b.

FIG. 10 is a schematic illustration of a method for forming a five-levelmicrofluidic network structure including a straight channel surroundedby a coiled series of interconnected channels;

FIG. 11 is a schematic illustration of a pattern on a material surfaceformed with a microfluidic stamp provided according to the invention;

FIG. 12 a is a perspective view of a schematic illustration of a lowerand an upper mold master for forming a basketweave microfluidic networkstructure provided by the invention;

FIGS. 12 b-12 c provide photocopies of photomicrographs of amicrofluidic network characterized by a network of channels arranged ina basketweave configuration in accordance with one embodiment of thepresent invention;

FIG. 12 d is a photocopy of an SEM image of a micromolded structureproduced according to one embodiment of the invention;

FIG. 13 is a photocopy of a photomicrograph of a microfluidic networkcomprising a straight channel surrounded by a coiled fluid flow pathcomprising a series of interconnected channels, according to oneembodiment of the invention;

FIG. 14 a is a schematic illustration of a microfluidic stamping processaccording to one embodiment of the invention;

FIG. 14 b is a schematic illustration of the fluid flow path layout ofthe microfluidic stamp illustrated in FIG. 14 a;

FIG. 14 c is a photocopy of a photomicrograph of a patterned surfaceproduced using the microfluidic stamp illustrated in FIG. 14 a;

FIG. 15 a is a schematic illustration of the layout of fluid flow pathsin one embodiment of a microfluidic stamp provided according to theinvention;

FIG. 15 b is a photocopy of photomicrograph of a stamped pattern on amaterial surface produced using a microfluidic stamp having themicrofluidic network structure illustrated in FIG. 15 a;

FIG. 16 a is a schematic illustration of the layout of fluid flow pathsin one embodiment of a microfluidic stamp provided according to theinvention;

FIGS. 16 b-16 d are photocopies of photomicrographs of patterned cellson a material surface deposited using a microfluidic stamp having themicrofluidic network configuration illustrated in FIG. 16 a;

FIG. 17 a is a schematic illustration of the layout of fluid flow pathsin one embodiment of a microfluidic stamp provided according to theinvention; and

FIGS. 17 b-17 e are photocopies of photomicrographs of patterned cellson a material surface deposited using a microfluidic stamp having themicrofluidic network configuration illustrated in FIG. 17 a.

DETAILED DESCRIPTION

The present invention is directed to fabrication methods for producingthree-dimensional microfluidic network structures, polymericmicrofluidic network structures having a three-dimensional array ofchannels included therein, and various uses for the microfluidicnetworks, for example as a template for forming and depositing complexpatterns on substrates. A “three-dimensional microfluidic network,”“three-dimensional microfluidic network structure,” or“three-dimensional microfluidic stamp” as used herein refers to astructure capable of containing a fluid and/or providing fluid flowtherethrough, which includes at least three channels therein, and maycontain many more; furthermore, the structure includes at least threechannels that are arranged with respect to each other such that thereexists no plane, or curved planar surface, which contains disposedtherein the longitudinal axes of the three channels. The microfluidicnetworks provided according to the invention, because of theirthree-dimensionality of structure, are able, for example, to providechannels within the structure having longitudinal axes (defined as theaxial centerline of the channel aligned parallel to the direction ofbulk fluid flow within the channel) aligned along each of the x, y, andz directional components of space. The ability to produce microfluidicstructures having channels arranged in a three-dimensional networkenables the systems provided according to the invention to includetherein a plurality of channels providing one or more independent fluidflow paths, where the channels and flow paths can be arrayed inarbitrarily complex geometric networks since the channels of thestructures have the capability of crossing over and/or under each otherwithin the structure.

One way to analogize the capabilities of the microfluidic networks, andmethods for producing the microfluidic networks, according to theinvention, is to compare the channel systems of the microfluidicnetworks to a knot in three-dimensional space. The microfluidic networksprovided according to the invention have the ability to fabricate thephysical realization of knots, and thus can include channel systems ofarbitrary topological complexity. In mathematical terms, a knot is aclosed, non-intersecting, curved line in three dimensions. Knots aretypically described in mathematics in terms by their projections onto aplane. For non trivial knots, these projections contain “double points”,which are points where the projected curve crosses itself. A knot canalways be slightly perturbed in three dimensions so that, in projection,it has no triple or higher order points: that is, points where theprojected curve crosses itself three or more times. Hence, knots can bedescribed completely by giving such a two-dimensional projection,together with information about which piece of the curve crosses over orunder the another piece at each double point.

The microfluidic networks provided according to the invention, becauseof their three-dimensional channel network structure, are able toprovide a physical realization of the above-mentioned double point. Inother words, the structures enable one channel, comprising a flow pathor a segment of a flow path, to cross over or under another channelproviding another flow path, a segment of another flow path, orproviding another segment of the same flow path. Thus, the inventivemicrofluidic networks can provide a physical realization of essentiallyany topological knot system. Likewise, the inventive networks canprovide a physical realization of essentially any arrangement ofinterlinked knots and of arbitrarily complex three-dimensional networksof interconnected channels whose projections onto a plane or surface, asexplained in more detail below, can contain any arbitrary number ofcrossings. As shown and explained in more detail below, in order for theinventive microfluidic networks to avoid intersection of channels attheir points of crossing in the planar projection, there typically areprovided at least three identifiable “levels” within the structure: a“lower” level that contains a channel disposed therein that crosses“under” an “upper” level that contains disposed therein a channel thatcrosses “over” the channel contained in the bottom level, and anintermediate level that isolates the channels of the lower and upperlevels and contains connecting channels penetrating therethrough thatfluidically connect the channels in the lower level and the channels onthe upper level in order to form a fluid flow path comprised of a seriesof interconnected channels. It should be understood that the terms“lower” and “upper” in the present context are intended to suggest onlythe relative positions of the various levels of the structure and arenot meant to imply any particular orientation of the structure in space.For example the structure can be flipped, rotated in space, etc. so thatthe “lower” level is positioned above the “upper” level or the levelscan be positioned side by side, etc. In yet other embodiments involvingflexible structures, the structure can be twisted or bent therebydeforming planar levels into curved surfaces in space such that the“upper” and “lower” levels of the structure may be positioneddifferently with respect to each other at different locations in theoverall structure. In order to produce microfluidic networks witharbitrarily complex channel networks, no additional levels are typicallyneeded because triple, or higher order points in the projection are notnecessary to allow the channels within the structure to cross over orunder each other and thus cross each other in space without physicalintersection of the “crossing” channels within the structure.

FIG. 1 a illustrates one exemplary embodiment of an essentially infinitenumber of microfluidic network structures that can be produced accordingto the invention. Microfluidic network structure 100 includes a seriesof interconnected channels providing seven non-fluidicallyinterconnected fluid flow paths. The channels are arranged in a “basketweave” arrangement. Channel system 100, as illustrated, includes threenon-fluidically interconnected fluid flow paths, 102, 104, and 106arrayed within planes parallel to the y-z coordinate plane, and fournon-fluidically interconnected flow paths 108, 110, 112, and 114 arrayedwithin planes parallel to the x-z coordinate plane. Each fluid flow pathof the structure comprises a series of interconnected channels (e.g.fluid flow path 102 comprises interconnected channels 113, 124, 126,116, 118, 120, 128, 122 and 123 within structure 100).

Flow path 102, for example, includes two channels 116 and 122 disposedwithin the first, lower level of structure 100 and two channels 120 and124 disposed within the second, upper level of the structure. Flow path102 also includes a number of connecting channels, e.g. 118, 126, and128 traversing a third, intermediate level of the structure andinterconnecting channels contained in the first, lower level and second,upper level of the structure. The microfluidic network provided bystructure 100 is truly three-dimensional because it cannot be producedby a two-dimensional structure comprising a series of interconnectedchannels disposed within a single plane or any stack or array of suchstructures. In other words, network 100 includes channels disposedwithin the first, lower level of the structure that are non-parallel tochannels disposed within the second, upper level of the structure (e.g.channel 116 of fluid flow path 102 and channel 130 of fluid flow path110). Another way to describe the three-dimensionality of network 100,and distinguish the network from those realizable in two-dimensionalsystem, is to point out, that, for example, flow path 102 comprises aseries of non-colinear interconnected channels disposed within a firstplane of the structure, which is parallel to the y-z coordinate plane,and a second fluid flow path, for example, fluid flow path 108, isdisposed within a second plane (parallel to the x-z coordinate plane asshown) that is not parallel with the first plane. Yet another way inwhich the microfluidic networks provided according to the inventiondiffer from those realizable with two-dimensional systems is that theinventive microfluidic systems can include a fluid flow path thereinhaving a longitudinal axis, defining a direction of bulk fluid flowwithin the flow path, that is not disposed within any single plane inspace, nor is disposed within any a surface that is parallel to anysurface (such as surface 132 or 134) of the microfluidic structure.

A “level” of a structure, as used herein, refers to a plane or curvedsurface within the structure, typically parallel to a top surface and abottom surface of the structure, which can have a channel or series ofchannels disposed therein and/or penetrating therethrough. It should beunderstood that in the discussion and figures illustrated below, themicrofluidic network structures are generally shown as having planarsurfaces (e.g. surfaces 132 and 134), such that the levels within thestructure are planar; however, many of the structures, as described inmore detail below, are fabricated from flexible and/or elastomericmaterials that are capable of being bent, twisted, or distorted from theillustrated planar configurations. For such embodiments, the “levels”within the structure will comprise curved surfaces that are parallel tothe distorted planar surfaces of the structure, and any discussionherein with regard to “levels” of the structures should be understood toencompass such curved surfaces as well as the planar surfacesillustrated. “Parallel,” when used in the context of comparing thetopology of two surfaces in space, has its common mathematical meaningreferring to the two surfaces being everywhere spaced apart from eachother equidistantly.

“Non-fluidically interconnected” fluid flow paths, as used herein,refers to fluid flow paths each comprising one channel or multiple,fluidically interconnected channels, where the channels of differentflow paths do not intersect and are physically isolated from each otherwithin the structure so that they can not communicate fluid between eachother through bulk mixing of fluid streams. A “fluid flow path” as usedherein refers to one channel or a series of two or more interconnectedchannels providing a space within the microfluidic structure able tocontain fluid or through which fluid can continuously flow. Each fluidflow path of the structure includes at least one opening thereto able tobe placed in fluid communication with the environment external to themicrofluidic structure and some preferred embodiments of fluid flowpaths include at least two openings able to be placed in fluidcommunication with the environment external to the microfluidicstructure, thus providing an inlet and an outlet. A “channel” as usedherein refers to a flow path or continuous segment of a flow path, whichis disposed within one or more levels of the microfluidic networkstructure and/or penetrates through one or more levels of themicrofluidic network structure. “Interconnected channels,” as usedherein, refers to two or more channels within the structure that areable to communicate fluid between and through each other. A “non-linear”flow path and/or channel, as used herein, refers to such flow path orchannel having a longitudinal axis that deviates from a straight linealong its length by more than an amount equal to the minimumcross-sectional dimension of the channel or flow path. A “longitudinalaxis” of a channel or flow path as used herein refers to an axisdisposed along the entire length of such channel or flow path, which iscoextensive with and defined by the geometric centerline of thedirection of any bulk fluid which would flow through the channel or flowpath should such channel or flow path be configured for fluid flowtherethrough. For example, a linear or “straight” channel would tend tohave a longitudinal axis that is essentially linear, while a fluid flowpath comprising a series of such straight channels that are fluidicallyinterconnected can have a longitudinal axis, comprising theinterconnected longitudinal axes of the individual interconnectedchannels forming the fluid flow path, which is “non-linear.” A channelwhich is “disposed within,” “disposed in,” “contained within,” or“contained in” a level or multiple levels of the structure refers hereinto such channel having a longitudinal axis that is coplanar with or, inthe case of a level defined by a curved surface, is lying along acontour of the surface, of the level(s) in which it is disposed orcontained. A channel that “penetrates,” “penetrates through,” or“traverses” a level or multiple levels of the structure refers herein tosuch channel having a longitudinal axis that is non-coplanar with or, inthe case of a level defined by a curved surface, is not lying along acontour of the surface of the level(s) such that the longitudinal axisof such channel is non-parallel with any line that can be disposedwithin the level.

Fluid flow path 102 of microfluidic network 100 communicates with theexternal environment through an inlet opening 136 in fluid communicationwith bottom surface 134 and an outlet opening 138 in fluid communicationwith upper surface 132. The other fluid flow paths of the network havesimilar inlet and outlet openings, as illustrated.

The channels of the microfluidic networks provided according to theinvention have at least one cross-sectional dimension that does notexceed about 500 μm, in other embodiments does not exceed about 250 μm,in yet other embodiments does not exceed about 100 μm, in otherembodiments does not exceed about 50 μm, and in yet other embodimentsdoes not exceed about 20 μm. A “cross-sectional dimension,” when used inthe above context, refers to the smallest cross-sectional dimension fora cross-section of a channel taken perpendicular to the longitudinalaxis of the channel. While the channels of network 100 havecross-sectional dimensions that are essentially equal to each other, inother embodiments, the channels can have unequal cross-sectionaldimensions, and some channels can have depths within the structuresufficiently great so that they are disposed in two or all three levelsof the structure, instead of being disposed in only a single level, asillustrated. In addition, while in network 100 the channels are straightand linear, in other embodiments the channels can be curved within thelevel(s) in which they are disposed.

The double points formed where the channels of the fluid flow paths ofnetwork 100 cross over each other are more clearly seen in thetwo-dimensional perpendicular projection shown in FIG. 1 b. FIG. 1 bshows microfluidic network 100 as projected onto the y-x plane as viewedin the negative z-axis direction. Crossover double point 140, forexample, represents the double point defining the cross over of channel130 of fluid flow path 110 and channel 116 of fluid flow path 102. Ingeneral, microfluidic networks provided according to the inventionhaving fluid flow paths including channels that “cross over” each otherrefers to structures including channel networks wherein a perpendicularprojection of the channels onto a surface defining a level of thestructure, in which either of the channels are disposed, at leastpartially overlap each other. A “perpendicular projection” refers to aprojection in a direction that is perpendicular or normal to the surfacebeing projected upon. “At least partially overlap” or “at leastpartially overlapping,” as used herein when referring to projections ofchannels which cross over each other, refers to the two-dimensionalprojection of the channels intersecting each other, as shown by point140 in FIG. 1 b, or, if, for example, the channels are arranged in aparallel direction with respect to each other within the networkstructure, to their being at least partially superimposed upon eachother in the two-dimensional projection.

While the three-dimensional microfluidic network structures describedherein could potentially be fabricated via conventionalphotolithography, microassembly, or micromachining methods, for example,stereolithography methods, laser chemical three-dimensional writingmethods, or modular assembly methods, as described in more detail below,the invention also provides improved fabrication methods for producingthe inventive structures involving replica molding techniques forproducing individual layers which comprise one or more of the levels ofthe structures, as discussed above. As described in more detail below,such layers are preferably molded utilizing mold masters having variousfeatures on their surface(s) for producing channels of the structure. Insome preferred embodiments, the features are formed via aphotolithography method, or can themselves comprise a molded replica ofsuch a surface.

The microfluidic network structures produced by the inventive methodsdescribed herein can potentially be formed from any material comprisinga solid material that comprises a solidified form of a hardenableliquid, and, in some embodiments, the structures can be injection moldedor cast molded. As will be described in more detail below, preferredhardenable liquids comprise polymers or precursors of polymers, whichharden upon, or can be induced to harden during, molding to producepolymeric structures. For reasons described in more detail below,particularly preferred polymeric materials for forming the microfluidicnetworks according to the invention comprise elastomeric materials.

For structures produced according to the preferred methods describedherein, the microfluidic networks provided according to the inventionwill typically be comprised of at least one discrete layer of polymericmaterial, and other embodiments will be comprised of at least twodiscrete layers of polymeric material, and in yet other embodiments willbe comprised of three or more discrete layers of polymeric material. A“discrete layer” of material as used herein refers to a separatelyformed subcomponent structure of the overall microfluidic structure,which layer can comprise and/or contain one, two, or three, or morelevels of the overall channel network of the microfluidic structure. Asdescribed and illustrated in more detail below, the discrete layers ofthe structure can be stacked together to form a three-dimensionalnetwork, or multiple three-dimensional networks, if desired, and canalso be, in some embodiments, placed between one or more support layersor substrate layers in order to enclose and fluidically seal channels ofthe lower and upper levels of the microfluidic structure.

As described in more detail below, the methods for producingmicrofluidic network structures provided by the invention can, in someembodiments, produce discrete layers comprising a single level of theoverall structure, wherein the three-dimensional network structure isformed by forming a first layer including a series of channels disposedtherein, forming a second layer including a second series of channelsdisposed therein, and forming a third layer having connecting channelstraversing the layer, and subsequently stacking the third layer betweenabove-mentioned first and second layers and aligning the layers withrespect to each other to achieve the overall desired three-dimensionalnetwork structure. In another embodiment, the microfluidic networkstructure includes two channel-containing layers: a first discrete layercontaining both a first level, including a series of channels disposedtherein, and a third, intermediate level of the structure including theconnecting channels traversing the level; and a second discrete layerincluding the second level of the structure, having a second series ofchannels disposed therein. In such a method the first discrete layer andthe second discrete layer are stacked and aligned with respect to eachother to produce the overall desired three-dimensional microfluidicnetwork structure. And in yet a third embodiment, all three levels ofthe microfluidic network structure can be produced in a single discretelayer, the layer comprising a three-level microfluidic membranestructure.

FIGS. 2 a and 2 b illustrate a microfluidic structure 150 having analternative three-dimensional arrangement of channels therein.Microfluidic network 150 includes two non-fluidically interconnectedflow paths 152 and 154. Fluid flow path 152 comprises a series ofinterconnected channels 156, 158, 160, 162 and 164, which are non-linearand which define a plane parallel to the y-z coordinate plane. Channels156 and 164 are disposed within a first, lower level of the structure,and channel 160 is disposed within a second, upper level of thestructure. Connecting channel 158 traverses a third, intermediate levelof the structure from the first, lower level to the second, upper leveland fluidically interconnects channel 156 to channel 160. Similarly,connecting channel 162 traverses the third, intermediate level of thestructure connecting channel 164 and channel 160. Flow path 152 isconnected in fluid communication with the external environment via inletopening 168 in side wall 170 an outlet opening 172 in side wall 174.Fluid flow path 154 comprises a single channel 176 disposed within thefirst, lower level of the structure, and is interconnected to theenvironment via inlet opening 178 in side wall 180 an outlet opening 182in side wall 190. The perpendicular projection of the microfluidicchannel network, onto the first, lower level of the structure isillustrated in FIG. 2 b. FIG. 2 b shows double point 192 where channel160 of fluid flow path 152 crosses over channel 176 of fluid flow path154.

FIGS. 3 a and 3 b illustrate yet another simple microfluidic networkprovided according to the invention but not achievable with aconventional two-dimensional microfluidic network structure.Microfluidic network 200 includes a single fluid flow path 202. Fluidflow path 202 is comprised of a first channel 204 disposed within afirst, lower level of the structure; a second channel 206 disposedwithin a second, upper level of the structure; and a connecting channel208 traversing a third, intermediate level of the structure andfluidically interconnecting channels 204 and 206. Channel 204 disposedwithin the first level of the structure and channel 206 disposed withinthe second level of the structure are non parallel to each other and, inthe illustrated embodiment, happen to be perpendicular to each other.FIG. 3 b illustrates the perpendicular projection of microfluidicnetwork 200 onto the first, lower level structure along the negativez-axis direction. As illustrated, microfluidic network 200 does notinclude any crossover points in the projection.

As previously discussed, a microfluidic network need only include threelevels therein (a first and a second level including channels disposedtherein such that their longitudinal axes are coplanar with a surfacedefining the level and a third intermediate level having one or moreconnecting channels passing therethrough fluidically connecting thechannels of the first level and the second level) in order to provideany arbitrarily complex network of channels that pass over and under oneanother. However, certain potentially desirable geometric configurationsof channels may require more than the three levels contained within thestructures discussed and illustrated above. For example, if it isdesired to produce a microfluidic network having channels disposedwithin three or more non-coplanar levels of the structure, additionallevels are needed. In general, the number of levels required formicrofluidic structures produced according to the invention required toproduce n levels, each level having channels disposed therein such thattheir longitudinal axis are coplanar with the level, requires a total of2n−1 total levels in the structure. Thus, for the previously illustratedembodiments having two levels therein in which channels are disposed,each structure requires a total of three levels to form the overallnetwork structure (an upper and lower level in which the channels aredisposed and an intermediate level through which the connecting channelspass).

FIGS. 4 a and 4 b illustrate one embodiment of a microfluidic structure,producible according to the methods of the invention described below,including therein three levels having channels disposed therein suchthat their longitudinal axes are coplanar with each of the levels, and atotal of five levels overall. Structure 220 includes a microfluidicnetwork comprising a fluid flow path 222 arranged as a coil surroundinga second fluid flow path 224. Such an arrangement may be especiallyuseful for particular microfluidic applications involving, for example,heat transfer or mass transfer between components contained within fluidflow paths 222 and 224, or for embodiments where electrical, magnetic,optical or other environmental interaction between materials in therespective flow paths is desired.

The first, lower level of structure 220 includes disposed thereinchannels 226, 228, 230, and 232 of coil flow path 222. The second levelfrom the bottom of structure 220 includes disposed therethrough thelowermost region 234 of connecting channels 236, 238, 240, 242, 244,246, and 248 of fluid flow path 222. The third level from the bottom ofstructure 220 includes channel 250 of fluid flow path 224 disposedtherein and also includes intermediate region 251 of the connectingchannels. The fourth level from the bottom of structure 220 includes,traversing therethrough, upper regions 252 of the connecting channels,and the uppermost level of structure 220 includes disposed thereinchannels 254, 256, 258 and 260 of flow path 222.

FIG. 4 b illustrates the perpendicular projection of microfluidicnetwork 220 onto a surface coplanar with the first, lowermost level ofthe structure that is parallel to the y-x coordinate plane, as viewed inthe negative z direction. As illustrated, structure 220 includes 8double point crossovers 264, 266, 267, 268, 269, 270, 272, and 274 whereeither flow path 224 crosses over a channel of flow path 222 (e.g.crossover points 264, 267, 269, and 272), or where channel 250 of flowpath 224 crosses under a channel of fluid flow path 222, (for example,crossover point 266, 268, 270, and 274.) It should be evident that thefive level structure illustrated by structure 220, in alternativeembodiments, can have flow paths therein comprising a series ofinterconnected channels arranged so as to yield higher order crossoverpoints than the double points illustrated. For example, in otherembodiments, a five level structure can have channels disposed thereinincluding triple point crossovers wherein a perpendicular projectiononto a surface coplanar with a level of the structure includes pointswhere three levels of channels intersect (i.e., where a channel disposedin the lowermost level, a channel disposed in the third, intermediatelevel, and a channel disposed in the uppermost level overlap and/orintersect each other in the two-dimensional projection).

As discussed above, the present invention also provides a variety ofmethods providing relatively simple and low cost fabrication techniquesfor producing the inventive microfluidic structures described herein.The preferred methods provided according to the invention and describedbelow are based upon utilizing a hardenable liquid to create replicamolded structures that comprise, or are assembled with other replicamolded structures to form, the three-dimensional microfluidic networkstructures provided by the invention.

FIGS. 5 a-5 c illustrate a first embodiment of a method for forming theinventive microfluidic structures by utilizing a replica molding processprovided by the invention. The method illustrated by FIGS. 5 a-5 cinvolves forming a number of replica molded layers from a hardenableliquid, each of which structures comprises a single level of the overallmicrofluidic network. Following the fabrication of each of the replicamolded structures comprising layers of the overall microfluidic networkstructure, the layers are stacked upon each other, aligned with respectto each other so that the respective molded features in the layerscreate the desired and predetermined microfluidic network pattern, and,optionally, the layers can be permanently sealed to each other and/or toone or more substrate layers, which substrate layers do not comprise alevel of the overall microfluidic structure, in order to yield afinished microfluidic network structure having a desired configuration.

Step 1 as illustrated in FIG. 5 a involves forming a first layer of thestructure comprising, for example, a first, lower level of themicrofluidic network. Of course, in other embodiments, layers comprisingan upper or intermediate level of the structure can be molded before orat the same time a lower layer is molded. In general, the order of themolding steps is not particularly critical and the various layers of theoverall structure can be molded in any order that is desired orconvenient. In the illustrated embodiment, a lower mold master 300 isprovided having a series of topological features 302 protruding from anupper surface 304 of the lower mold master. A second mold master 306having a flat, featureless surface 308 facing surface 304 of mold master300 is provided and placed in contact with an upper surface oftopological features 302 of mold master 304. Disposed between moldmasters 304 and 306 is a layer of hardenable liquid 310, which uponsolidification forms a replica molded layer including therein aplurality of channels, formed by topological features 302 of mold master304, which, channels, in preferred embodiments, pass completely throughthe thickness of the entire layer of liquid 310, upon hardening, thusforming a membrane structure comprised of the hardened liquid.

Mold master 300, having positive, high-relief topological features 302formed on a surface 304 thereof comprises, in some preferredembodiments, a substrate that has been modified, for example, viaphotolithography or any suitable micromachining method apparent to thoseof ordinary skill in the art. Topological features 302 are shaped,sized, and positioned to correspond to a desired arrangement of channelsin the level of the overall microfluidic network structure being formedby the mold master. In one preferred embodiment, mold master 300comprises a silicon wafer having a surface 304 that has been viaphotolithography utilizing a photomask having a pattern therewithincorresponding to a desired pattern of topological features 302.Techniques for forming positive relief patterns of topological featureson silicon, or other materials, utilizing photolithography andphotomasks, are well known and understood by those of ordinary skill inthe art and, for example, are described in Qin, D., et al. “RapidPrototyping of Complex Structures with Feature Sizes Larger Than 20microns,” Advanced Materials, 8(11):pp.917-919 and Madou, M.,Fundamentals of Microfabrication, CRC Press, Boca Raton, Fla., (1997),both incorporated herein by reference.

In a particularly preferred embodiment, mold master 300 comprises asilicon or other substrate, which has been spincoated with one or morelayers of a commercially available polymeric photoresist material. Insuch preferred embodiments, topological features 302 can be easily,conveniently, and accurately formed in the layer(s) of photoresistforming surface 304 of substrate 300 via exposure of photoresist toradiation through a photomask and subsequent development of thephotoresist material to remove photoresist material from the surface andregions surrounding features 302 thus leaving behind topologicalfeatures 302 in positive relief. A variety of positive and negativephotoresists can be utilized for such purposes and are well known tothose of ordinary skill in the art.

One particularly preferred method for forming topological features 302on a surface of a substrate coated with one or more layers ofphotoresist is described in more detail below in the context of FIG. 8.The photomask utilized, as described above, provides a pattern thereinable to selectively block radiation reaching the layer(s) of photoresistso that, upon development of the layer, a pattern of topologicalfeatures will be formed, which features correspond to a desiredarrangement of channels within the replica molded layer. Such patternscan be designed with the aid of any one of a number of commerciallyavailable computer aided design (CAD) programs, as would be apparent tothose of ordinary skill in the art.

Mold master 306 can be comprised of the same material as mold master300; however, in preferred embodiments, mold master 306 is formed of anelastomeric material, for example, an elastomeric polymer. Mold master306 is, in preferred embodiments, formed of an elastomeric materialbecause the elastomeric nature of the mold master enables an improvedseal at the interface of surface 308 of mold master 306 and the uppersurfaces of topological features 302 of lower mold master 300 to beformed so as to essentially completely exclude hardenable liquid 310from the interface between the topological features 302 and surface 308of mold master 306. This preferred (“sandwich”) method enables, upon thehardening of hardenable liquid 310, the production of a membranecomprised of the hardened fluid having channels disposed therein whichcompletely traverse the entire thickness of the membrane and which arenot blocked by a thin layer of hardened liquid.

For some embodiments, it is also desirable that upper mold master 306 betransparent in order to be able to visualize topological features 302during the molding process. Alternatively, in other embodiments, uppermold master 306 can comprise a rigid, non-elastomeric material and lowermold master 300, including topological features 302 forming the channelsof the molded structure, can be formed of an elastomeric material. Insuch an embodiment, the elastomeric mold master having positive relieftopological features disposed on its surface is preferably itself formedas a molded replica of a pre-master having a surface including aplurality of negative, low-relief features therein, which form thepositive relief features in the elastomeric mold master upon creating areplica mold of the pre-master surface. In yet other embodiments, theupper and lower mold masters of the invention can both compriseelastomeric materials and can be formed of the same, or differentelastomeric materials. In addition, although less preferred, upper moldmaster 306 can be eliminated entirely and hardenable fluid 310 maysimply be spuncast onto surface 304 of lower mold master 300 to athickness corresponding to the height of topological features 302. Suchmethod is generally less preferred for producing molded membranesaccording to the invention because it is generally desired that theuppermost and lowermost surfaces of the membrane be as flat and smoothas possible to enable conformal sealing and prevention of leakage uponassembly of the layers into the overall microfluidic network structure.

In preferred embodiments, hardenable liquid 310 is placed upon surface304 of lower mold master 300 in an amount sufficient to form a layerover the region of surface 304 including topological features 302,corresponding to the channel structure in the layer to be formed, whichlayer having a thickness at least equal to the height of topologicalfeatures 302 above surface 304. Subsequent to placing liquid 310 onsurface 304, the method involves bringing surface 308 of upper moldmaster 306 into contact with the upper surface of features 302. Inalternative embodiments, a lower mold master and upper mold master canbe brought into contact prior to addition of the hardenable liquid, andthe hardenable liquid can be applied to the region between the facingsurfaces of the mold masters by adding a sufficient amount in the regionof the space between the upper mold master and lower mold master aroundtheir periphery (e.g. periphery 312), and subsequently allowinghardenable liquid 310 to flow into the space surrounding the topologicalfeatures of the mold master(s) via capillary action. Such method forutilizing capillary action for creating a molded replica structure asdescribed in detail in commonly owned, copending U.S. patent applicationSer. No. 09/004,583 entitled “Method of Forming Articles IncludingWaveguides Via Capillary Micromolding and Microtransfer Molding,” andInternational Pat. Publication No. WO 97/33737, each incorporated hereinby reference.

Hardenable liquid 310 can comprise essentially any liquid known to thoseof ordinary skill in the art that can be induced to solidify orspontaneously solidifies into a solid capable of containing andtransporting fluids contemplated for use in and with the microfluidicnetwork structures. In preferred embodiments, hardenable liquid 310comprises a polymeric liquid or a liquid polymeric precursor (i.e. a“prepolymer”). Suitable polymeric liquids can include, for example,thermoplastic polymers, thermoset polymers, or mixture of such polymersheated above their melting point; or a solution of one or more polymersin a suitable solvent, which solution forms a solid polymeric materialupon removal of the solvent, for example, by evaporation. Such polymericmaterials, which can be solidified from, for example, a melt state or bysolvent evaporation, are well known to those of ordinary skill in theart.

In preferred embodiments, hardenable liquid 310 comprises a liquidpolymeric precursor. Where hardenable liquid 310 comprises aprepolymeric precursor, it can be, for example, thermally polymerized toform a solid polymeric structure via application of heat to mold master300 and/or mold master 306; or, in other embodiments, can bephotopolymerized if either mold master 300 or mold master 306 istransparent to radiation of the appropriate frequency. Curing andsolidification via free-radical polymerization can be carried out aswell. These and other forms of polymerization are known to those ofordinary skill in the art and can be applied to the techniques of thepresent invention without undue experimentation. All types ofpolymerization, including cationic, anionic, copolymerization, chaincopolymerization, cross-linking, and the like can be employed, andessentially any type of polymer or copolymer formable from a liquidprecursor can comprise hardenable liquid 310 in accordance with theinvention. An exemplary, non-limiting list of polymers that arepotentially suitable include polyurethane, polyamides, polycarbonates,polyacetylenes and polydiacetylenes, polyphosphazenes, polysiloxanes,polyolefins, polyesters, polyethers, poly(ether ketones), poly(alkalineoxides), poly(ethylene terephthalate), poly(methyl methacrylate),polystyrene, and derivatives and block, random, radial, linear, orteleblock copolymers, cross-linkable materials such as proteinaceousmaterials and/or blends of the above. Gels are suitable wheredimensionally stable enough to maintain structural integrity uponremoval from the mold masters, as described below. Also suitable arepolymers formed from monomeric alkylacrylates, alkylmethacrylates,alpha-methylstyrene, vinyl chloride and other halogen-containingmonomers, maleic anhydride, acrylic acid, acrylonitrile, and the like.Monomers can be used alone, or mixtures of different monomers can beused to form homopolymers and copolymers. The particular polymer,copolymer, blend, or gel can be selected by those of ordinary skill inthe art using readily available information and routine testing andexperimentation so as to tailor a particular material for any of a widevariety of potential applications.

According to some preferred embodiments of the invention, hardenableliquid 310 comprises a fluid prepolymeric precursor which forms anelastomeric polymer upon curing and solidification. A variety ofelastomeric polymeric materials are suitable for such fabrications, andare also suitable for forming mold masters, for embodiments where one orboth of the mold masters is composed of an elastomeric material. Anon-limiting list of examples of such polymers includes polymers of thegeneral classes of silicone polymers, epoxy polymers, and acrylatepolymers. Epoxy polymers are characterized by the presence of athree-membered cyclic ether group commonly referred to as an epoxygroup, 1, 2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Examples of silicone elastomerssuitable for use according to the invention include those formed fromprecursors including the chlorosilanes such as methylchlorosilanes,ethylchlorosilanes, and phenylchlorosilanes, and the like. Aparticularly preferred silicone elastomer is polydimethylsiloxane(PDMS). Exemplary polydimethylsiloxane polymers include those sold underthe trademark Sylgard by Dow Chemical Co., Midland, Mich., andparticularly Sylgard 182, Sylgard 184, and Sylgard 186.

Silicone polymers, for example, PDMS, are especially preferred for usein the invention because they have several desirable beneficialproperties simplifying fabrication of the microfluidic networkstructures, described herein. First, such materials are inexpensive,readily available, and can be solidified from a prepolymeric liquid viacuring with heat. For example, PDMSs are typically curable by exposureof the prepolymeric liquid to temperatures of about, for example, 65° C.to about 75° C. for exposure times of about, for example, 1 hour.Second, silicone polymers, such as PDMS, are elastomeric and are thususeful for forming certain of the mold masters used in some embodimentsof the invention. In addition, microfluidic networks formed fromelastomeric materials can have the advantage of providing structureswhich are flexible and conformable to the shape of a variety ofsubstrates to which they may be applied, and elastomeric networks canprovide reduced resistance to fluid flow for a given applied pressuredrop, as compared to non-elastomenrc structures, and can also be moreeasily fabricated to include active elements therein, for exampleintegrated valves and pumping elements, which elements can utilize theflexibility and elasticity of the material for their performance.

Another distinct advantage for forming the inventive microfluidicnetworks from silicone polymers, such as PDMS, is the ability of suchpolymers to be oxidized, for example by exposure to an oxygen-containingplasma such as an air plasma, so that the oxidized structures contain attheir surface chemical groups capable of cross-linking to other oxidizedsilicone polymer surfaces or to the oxidized surfaces of a variety ofother polymeric and non-polymeric materials. Thus, membranes, layers,and other structures produced according to the invention utilizingsilicone polymers, such as PDMS, can be oxidized and essentiallyirreversibly sealed to other silicone polymer surfaces, or to thesurfaces of other substrates reactive with the oxidized silicone polymersurfaces, without the need for separate adhesives or other sealingmeans. In addition, microfluidic structures formed from oxidizedsilicone polymers can include channels having surfaces formed ofoxidized silicone polymer, which surfaces can be much more hydrophilicthan the surfaces of typical elastomeric polymers. Such hydrophilicchannel surfaces can thus be more easily filled and wetted with aqueoussolutions than can structures comprised of typical, unoxidizedelastomeric polymers or other hydrophobic materials.

In addition to being irreversibly sealable to itself, oxidized PDMS canalso be sealed irreversibly to a range of oxidized materials other thanitself including, for example, glass, silicon, silicon oxide, quartz,silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxypolymers, which have been oxidized in a similar fashion to the PDMSsurface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention are described in more detail below and also in Duffy et al.,Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane,Analytical Chemistry, Vol. 70, pages 474-480, 1998, incorporated hereinby reference.

For clarity and simplicity, the discussion below involving the inventivemethods for forming microfluidic structures according to the inventionin many instances makes specific reference to a preferred embodimentwherein the layers comprising the structure and/or one or more moldmasters are formed from a hardenable liquid comprising a fluidprepolymer of PDMS. It should be understood, as the discussion abovemakes clear, that such reference is pure exemplary, and a wide varietyof other materials can be utilized in place of or in addition to PDMS toachieve the various objects, features, and benefits of the presentinvention, as would be apparent to those of ordinary skill in the art.

Referring again to FIG. 5 a, in Step 2, PDMS, comprising hardenableliquid 310, is cured and solidified, for example by application of heatto raise the temperature of the PDMS prepolymer to between about 65° C.to about 75° C. for about 1 hour, as described above. In order toprevent seepage of the PDMS between surface 308 and the upper surface oftopological features 302, it is preferred to apply pressure to one orboth of lower surface 314 of mold master 300 and upper surface 316 ofmold master 306. It has been found, within the context of the invention,that a pressure of approximately between about 10-100 g/mm² (100-1,000kPa) or greater is generally sufficient to prevent PDMS prepolymer fromseeping between topological features 302 and surface 308 so as to causeblockage of subsequent channels formed within the cured membrane.

Step 3 involves peeling the cured membrane from one or both of moldmaster 300 and 306. In preferred embodiments, as discussed above,materials are selected for mold master 300, mold master 306, andhardenable liquid 310, which allow removal of the solidified membraneupon solidification of the hardenable liquid without destruction of themolded structure. In especially preferred embodiments, because asolidified layer is typically thin and fragile (for example, layer 318can vary in thickness from about 20 μm to about 1 mm), mold master 300and mold master 306 are selected or treated such that layer 318 adheresto the surface of one of the mold masters more strongly than to thesurface of the other mold master. Such differential adhesion allows themold masters to be peeled apart such that the fragile molded layer 318remains adherent to and is supported by one or the other of the moldmasters. Such differential adhesion of layer 318 can be created byselecting materials comprising mold master 306 and surface 304 of moldmaster 300 having different chemical properties such that thenon-covalent interfacial adhesion between layer 318 and surface 304differs from that between layer 318 and surface 308. Those of ordinaryskill in the art can readily determine appropriate materials forcomprising hardenable liquid 310, mold master 300, and mold mater 306and/or surface treatments which can be applied to either or both of themold masters that allow for differences in non-covalent interfacialadhesion between layer 318 and the surfaces of the mold masters,enabling layer 318 to be selectively removed from one of the surfaceswhile remaining adherent to the other. Interfacial free energies for awide variety of materials are readily available to those of ordinaryskill in the art and can be utilized, along with routine screeningtests, for example measuring forces required to peel apart variouscombinations of materials, by those of ordinary skill in the art toreadily select a combination of materials, without undueexperimentation, for enabling layer 318 to be selectively removed fromthe surface of one mold master while remaining adherent to and supportedby the surface of the other mold master.

For example, in the illustrated embodiment, lower mold master 300includes an upper surface 304 comprising a negative photopolymer(SU-8-50, Microlithography Chemical Corp., Newton, Mass.), upper moldmaster 306 comprises oxidized PDMS, and hardenable fluid 310 comprises aPDMS prepolymer. Also in the illustrated embodiment, surfaces 308 and304, before contact with fluid 310 were silanized to facilitate theremoval of PDMS replica layer 318 after curing. In an exemplaryembodiment, the masters were silanized by exposing the surfaces to achlorosilane vapor, for example a vapor containingtridecafluoro-1,1,2,2-tetrahydrooctal-1-trichlorosilane. PDMS replicalayer 318 adheres more strongly to silanized PDMS mold master 306 thanto silanized surface 304 of mold master 300 and remains supported by andattached to mold master 306 upon applying a peeling force tending toseparate the two mold masters, resulting in molded replica layer 318remaining adherent and supported by mold master 306, as illustrated inStep 3. In an alternative embodiment, instead of utilizing a silanizedPDMS layer for mold master 306 in combination with silanized mold master300, as described above, mold master 306 can comprise a layer or sheetof a material having a very low interfacial free energy, for exampleTeflon™ (polytetrafluoroethylene (PTFE)). In such an embodiment, replicamolded layer 318 will tend to remain adherent to mold master 300 uponapplying a peeling force tending to separate mold master 306 and moldmaster 300.

Step 4 of FIG. 5 a illustrates an optional step comprising conformallycontacting molded replica layer 318, supported by mold master 306, witha lower substrate layer 320, and, optionally, irreversibly sealing lowersurface 319 of layer 318 to the upper surface 322 of substrate 320. Inthe illustrated embodiment, substrate 320 comprises a PDMS slab having aflat upper surface 322. Both lower surface 319 of layer 318 and uppersurface 322 of substrate 320 have been oxidized, for example by exposureto an air plasma in a plasma cleaner, as discussed above and in moredetail below, prior to bringing the surfaces into contact, so that whenbrought into conformal contact, an irreversible seal spontaneously formsbetween surface 319 and surface 322 providing a fluid-tight seal at thebottom of channels 321 in layer 318. Exposure of the PDMS surfaces tothe oxygen-containing plasma is believed to cause the formation of Si—OHgroups at the surface of the PDMS, which react with other Si—OH groupsto form bridging, covalent siloxane (Si—O—Si) bonds by a condensationreaction between the two oxidized PDMS surfaces.

In alternative embodiments, where it is not desired to permanently seallayer 318 to substrate 320, the surfaces may not be oxidized so thatthey do not irreversibly seal to each other but rather may simply bebrought into conformal contact with each other, which conformal contactbetween the two essentially flat planar surfaces can be sufficient, formicrofluidic applications involving vacuum or low pressures, to form afluid-tight seal. Also, in some applications, such as microcontactsurface patterning with the inventive microfluidic networks as describedin more detail below, it may be desirable to provide a “patterning”surface of the microfluidic network having channels therein which arenot sealed by a substrate, and which can be brought into contact with amaterial surface in order to form on the surface a pattern defined bythe channels in the “patterning” surface of the microfluidic network.

In yet other embodiments, substrate 320 can comprise a materialdifferent from one or both of molded layer 318 and mold master 306, forexample, a material other than PDMS. In some such embodiments, substrate320 can comprise, for example, the surface of a silicon wafer ormicrochip, or other substrate advantageous for use in certainapplications of the microfluidic network provided according to theinvention. Molded layer 318 can, as described above, be irreversiblysealed to such alternative substrates or may simply be placed inconformal contact without irreversible sealing. For embodiments where itis desired to irreversibly seal a molded replica layer 318 comprisingPDMS to a substrate 320 not comprising PDMS, it is preferred thatsubstrate 320 be selected from the group of materials other than PDMS towhich oxidized PDMS is able to irreversibly seal (e.g., glass, silicon,silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxypolymers, and glassy carbon surfaces which have been oxidized). Forembodiments involving hardenable liquids other than PDMS prepolymers,which form molded replica layers not able to be sealed via the oxidationmethods described above, when it is desired to irreversibly seal suchlayers to each other or to a substrate, alternative sealing means can beutilized, as would be apparent to those of ordinary skill in the art,including, but not limited to, the use of separate adhesives, thermalbonding, solvent bonding, ultrasonic welding, etc.

Step 5 illustrated in FIG. 5 a comprises the removal of upper moldmaster 306 to expose flat, top surface 317 of molded replica 318 thusyielding a first, lower level of the overall microfluidic networkstructure having a series of channels 321 disposed in a desired patterntherein. In an alternative embodiment to the illustrated membranesandwich method for forming membrane layer 318, in Step 1 a moldedreplica can be formed by placing mold master 300 in the bottom of a dishor other container having a depth in excess of the height of topologicalfeatures 302 and filling the container to a level in excess of theheight of features 302 with a hardenable liquid, such as PDMSprepolymer. Upon curing and removing the cured structured from thecontainer and from mold master 300, a structure similar to that obtainedat the end of Step 5 is formed, except comprising channels that do notpenetrate through the entire thickness of the molded replica layer. Suchan embodiment is described in further detail in the context of thefabrication method illustrated in FIG. 7 below. In addition, asillustrated in FIG. 5, to facilitate the stacking and alignment ofadditional molded replica layers comprising the second, third, and anyhigher levels of the microfluidic structure, lower layer 318 can betrimmed such that it is essentially uniform in thickness and has adesired overall size and perimeter shape.

FIG. 5 b illustrates the formation of a second molded replica layercomprising the third, intermediate level of a microfluidic networkstructure containing therein connecting channels as previouslydescribed. Steps 6-8 are essentially similar to Steps 1-3 describedabove in the context of FIG. 5 a, except that lower mold master 330 hasan upper surface 332 including thereon positive relief topologicalfeatures 334 protruding above surface 332 that are shaped, sized, andpositioned to form channels within the molded replica structurecorresponding to a desired arrangement of connecting channels within thethird, intermediate level of the microfluidic network structure beingfabricated. In addition, if desired, additional features (not shown) canbe included on the surface 332 of mold master 330 corresponding tochannels that are disposed within (i.e., have longitudinal axis coplanarwith) the third, intermediate level of the microfluidic networkstructure being formed.

Step 7 involves curing PDMS prepolymer 310 (or other hardenable liquid)as previously described for Step 2 above, and Step 8 involvesselectively removing molded replica layer 340 from mold master 330 whileit remains supported by an adherent to upper mold master 306, asdescribed for Step 3 above. Optional step 9 involves removing moldedreplica layer 340 from upper mold master 306 and, if desired, trimminglayer 340 so that it has an essentially identical overall size andperimeter shape as layer 318 above. Step 10 involves placing moldedreplica layer 340 into conformal contact with upper surface 317 ofmolded replica layer 318, aligning the channels 342 in molded replicalayer 340 with channels 321 in molded replica layer 318 to provide adesired registration between the channels of the first, lower level ofthe structure comprised of layer 318 and the third, intermediate levelof the structure comprised of layer 340, followed by irreversiblysealing together layers 318 and 340. In alternative embodiments, thealignment and sealing steps can be delayed if desired and performed inone step for all of the layers (i.e., all three channel-forming layers)comprising the overall structure which have been formed and stacked uponeach other (e.g. see FIG. 5 c below). In addition, for embodimentswherein upper mold master 306 is transparent, for example forembodiments where upper mold master comprises PDMS, and especially forembodiments including replica layers having a large number of channelsdisposed completely through the entire thickness of the membrane layeror having channels shaped so that the molded replica membrane layer isnot free-standing when removed from a support surface (e.g., channelscomprising continuous, closed geometric shapes, spiral shaped channels,etc.), layer 340 is preferably not removed from mold master 306 asillustrated in Step 9, but instead, mold master 306, with molded replicalayer 340 attached thereto, is placed in contact with upper surface 317of molded replica layer 318 and aligned and sealed as described in step10 prior to removing mold master 306, so that the molded replica layerremains attached to and supported by a mold master during each of themanipulation steps and is never free-standing.

Alignment of the molded replica features comprising the channels oflayers 318 and 340 can be accomplished utilizing a microscope, such as astereo microscope, in combination with an alignment stage and/ormicromanipulators for accurately positioning the layers and registeringthe features with respect to each other. For a preferred embodimentwherein layers 318 and 340 are comprised of PDMS, layers 318 and 340 canbe aligned and sealed to each other by either of the preferred methodsdescribed directly below. In a first method, layer 340 is placed uponlayer 318 and carefully aligned with respect to layer 318 to provide adesired alignment and registration of channels by utilizing a stereomicroscope and a micromanipulator. Layers 318 and 340 are then carefullyslightly separated from each other (e.g. by a few millimeters), withoutchanging the registered lateral alignment of channels within the layers,to provide a small space between surface 317 of layer 318 and surface341 of layer 340. The aligned structure having the layers slightlyseparated is then exposed to an oxygen-containing plasma in order tooxidize surfaces 317 and 341. The layers are then carefully broughttogether without altering or disturbing the lateral alignment of thechannels, so that surfaces 317 and 341 spontaneously seal to each otherupon conformal contact.

In the second, especially preferred, embodiment, alignment and sealingof the layers proceeds as follows. The upper surface 317 of layer 318and lower surface 341 of layer 340 are oxidized utilizing theoxygen-containing plasma exposure method described previously, and aliquid that is essentially non-reactive with the oxidized surfaces isplaced upon layer 317 to form a continuous layer of liquid thereupon,upon which, surface 341 of layer 340 is placed. The liquid, in additionto being essentially non-reactive with the oxidized surfaces of thePDMS, also preferably prevents degradation of the active Si—OH groupspresent on the surfaces for a period of time sufficiently long to enablealignment of the surfaces with respect to each other and removal of theliquid. After placing layer 340 onto the fluid-covered surface of layer318, layer 340 is aligned with respect to layer 318 to yield a desiredregistration and alignment of features (channels) for forming themicrofluidic network structure. The non-reactive liquid is then removedfrom between the two surfaces bringing the two surfaces into conformalcontact with each other and spontaneously sealing the two surfacestogether.

A variety of liquids can potentially be utilized as the non-reactiveliquid in the context of the inventive alignment method above described.As previously discussed, appropriate liquids will be essentiallynon-reactive with the oxidized surfaces and will preferably stabilizeand delay degradation of the active chemical groups contained within theoxidized surfaces. It has been found, in the context of the presentinvention, that polar liquids, and especially those comprising compoundsincluding hydroxyl moieties, are effective for use as the non-reactiveliquid. Especially preferred are water, alcohols, and mixtures thereofwith alcohols, and alcohol-water mixtures being particularly preferred,especially those including methanol and/or trifluoroethanol. Thenon-reactive liquid, in preferred embodiments, is removed from betweenthe oxidized surfaces of the layers via evaporation of the liquid, andthus, in such embodiments, as the non-reactive liquid evaporates theoxidized surfaces of the layers are simultaneously brought together inconformal contact whereupon the surfaces react to create an essentiallyirreversible seal.

While we have described above an embodiment wherein layer 340 comprisingthe third, intermediate layer of the structure is aligned and sealedwith respect to layer 318 comprising a first, lower level of thestructure prior to the fabrication of the molded replica layercomprising a second, upper level of the structure, in other embodiments,as mentioned above, the upper layer is formed prior to sealing the lowerand intermediate layers together, so that the intermediate and upperlayers can be stacked, aligned, and sealed to the lower layer in asingle step, eliminating the need to selectively oxidize only lowersurface 341 of intermediate layer 340 so as to prevent degradation of anoxidized upper surface 343 of intermediate layer 340 prior to theformation, stacking, and alignment of the upper layer to theintermediate layer (as shown and described in FIG. 5 c below).

FIG. 5 c illustrates the final steps for forming the overallthree-layer, three-level microfluidic network according to this firstfabrication method embodiment of the invention. Step 11 and Step 12 ofFIG. 5 c are analogous to Steps 1 and 2 of FIG. 5 a and Steps 6 and 7 ofFIG. 5 b and involve sandwiching a hardenable liquid 310, such as PDMS,between upper mold master 306 and a lower mold master 350 having anupper surface 352 including thereon topological features 354 in positiverelief constructed and positioned for forming channels disposed withinthe second, upper level of the final overall microfluidic networkstructure. Hardenable liquid 310 is cured and solidified in Step 12, aspreviously described, and, in preferred embodiments, molded replicalayer 360 is preferentially separated from surface 352 of lower moldmaster 350 while remaining in contact with and supported by upper moldmaster 306, as previously described. Molded replica layer 360, whichcomprises the second, upper level of the overall structure, includesmolded channels 362 disposed within layer 360. Step 14 involvesoptionally removing molded replica membrane layer 360 from upper moldmaster 306, as previously described for Step 9 discussed in the contextof FIG. 5 b. In step 15, molded replica layer 360, formed in Step 12above, is stacked upon intermediate layer 340, produced as described inthe context of FIG. 5 b above, and is subsequently aligned with respectto lower layers 340 and 318 such that channels 362 are registered andarranged in a desired alignment with respect to channels 342 of layer340 and channels 321 of layer 318 to provide a desired overallthree-dimensional fluidic network structure. Layer 360 is preferablysealed to layer 340 by utilizing one of the aligning and sealing methodspreviously described in the context of Step 10 of FIG. 5 b above.

As previously mentioned, in some preferred embodiments, layer 340 isaligned with respect to layer 318 and layer 360 is aligned with respectto layer 340 and the layers are sealed together in a single step afteralignment, which step, for such embodiments, can take place at Step 15of FIG. 5 c. In such embodiments, layer 340 would not be irreversiblysealed to layer 318 prior to the addition of layer 360 to the stack andalignment of layer 360 with respect to layer 340 and 318. In suchembodiments, wherein layers 340 and 360 are both aligned and sealed in asingle step, the alignment and sealing methods utilized can beessentially the same as those previously described for aligning andsealing layer 340 to layer 318 in the context of Step 10 of FIG. 5 b. Inaddition, in some embodiments where it is desired to irreversibly sealtogether some portions of the surfaces of the layers of the structureswhile leaving non-irreversibly sealed other portions, such portionswhich are not desired to be irreversibly sealed can be coated with aprotective coating (e.g. petroleum jelly) prior to oxidation in order toprevent oxidation of that portion of the surface so that it will notirreversibly seal to other oxidized surfaces upon contact.

Also provided, according to the invention, is a method for self-aligninglayers 318, 340, and 360 with respect to each other to provide a desiredalignment and registration of the channels within each of the layers,without the need for manual alignment with the aid of a microscopeand/or micromanipulator. The self-alignment method provided according tothe invention can be utilized for the embodiments described abovewherein the layers are oxidized and separated from each other by a layerof liquid during alignment of the layers. Details of this self-alignmentmethod are described below in the context of FIG. 6 and rely on theinteraction between the surface tension of the liquid between the layersand specific alignment features provided within the layers beingaligned.

Microfluidic network structure 370 obtained at the conclusion of Step 15of FIG. 5 c can comprise, for some embodiments, a complete structure,useful, for example, for applications wherein it is desired thatchannels 362 in layer 360 remain uncovered and exposed to thesurroundings. For example, one particular embodiment utilizing amicrofluidic network structure similar in configuration to structure 370involves utilizing the microfluidic network structure as a stampingtemplate for selectively applying a fluid to a material surface tocreate a pattern on the material's surface corresponding to the patternof channels 362 in layer 360. In such embodiments, surface 364 of layer360 comprises a stamping surface, which is placeable in contact with amaterial surface for forming a pattern thereon, and microfluidic networkstructure 370 comprises a three-dimensional microfluidic stamp. Specificuses and patterns producible by such microfluidic stamps are describedin greater detail below.

For other embodiments where it is desired to form a microfluidic networkstructure having an enclosed network of channels, optional Step 16 ofFIG. 5 c involves contacting upper surface 364 of layer 360 with anupper substrate layer 380 to form enclosed microfluidic networkstructure 390. In some preferred embodiments, where layers 318, 360, and364 comprise PDMS, upper substrate layer 380 is also comprised of PDMSand is irreversibly sealed to surface 364 via the self-sealing methodutilizing oxidation of the PDMS surfaces with an oxygen-containingplasma described in detail above. In alternative embodiments, however,upper substrate layer 380 may simply be placed in conformal contact withupper layer 364 and not irreversibly sealed thereto. In addition, uppersubstrate 380, in some embodiments, is not formed of the same material(e.g., PDMS) as layers 318, 360, and 364 of the structure. Uppersubstrate 380 can be essentially any of the materials mentionedpreviously for comprising substrate layer 320 previously described abovein the context of FIG. 5 a or any other substrate which can contactsurface 364 conformally.

In order to provide fluid communication between channels containedwithin layers 318, 360, and 364 of structure 390 and the surroundingenvironment, lower substrate layer 320 and/or upper substrate layer 380can include, formed therein, inlet/outlet conduits 392 providing fluidcommunication between the channels of the structure and the externalenvironment. Conduits 392 can be formed within substrate layer by avariety of machining and/or molding methods, as would be apparent tothose of ordinary skill in the art. In one embodiment, the conduits 392in substrate 320, comprising PDMS, are formed by carefully boring intolayer 320 with a small diameter syringe needle. In other embodiments,substrate layer 392 can itself comprise a replica molded structure withconduits 392 corresponding to and formed by topological features presenton a surface of a mold master utilized to form substrate layer 320. Inaddition, as would be apparent to those of ordinary skill in the art,other features can be machined within, or molded within one or both ofsubstrate layers 320 and 380 to provide various desired structures andfunctions for particular applications. For example, upper substratelayer 380 as shown includes traversing therethrough a small diameterchannel 394, having a characteristic cross sectional dimension on theorder of a few microns to a few tens of microns, which conduit 394serves the function of providing a relief valve to prevent over pressureof the channels contained within the structure defined by layers 318,340, and 360.

FIGS. 6 a-6 c illustrate one method for self-aligning various layers ofthe microfluidic network structures with respect to each other providedby the invention. The self-alignment method outlined in FIGS. 6 a-6 ccan be utilized for embodiments involving the alignment and sealingmethods discussed above involving disposing layers of the structureseparated from each other by a layer of liquid disposed therebetween.Such a method is useful, for example, for aligning layers 340 and 318with respect to each other and layers 360 and 340 with respect to eachother in the previously described microfluidic network fabricationmethod. In addition, the self-alignment method described in FIGS. 6 a-6c can also be utilized for performing self-alignment in the context ofthe methods described below in FIG. 7 and FIG. 10.

One embodiment for implementing the self-aligning method providedaccording to the invention is illustrated in FIG. 6 a. FIG. 6 a shows afirst layer 400 and a second layer 402 including therein replica moldedfeatures 404 and 406 respectively, comprising, for example, channelsdisposed within each of the layers, which channels are desired to beregistered and aligned with respect to each other in a certain way. Inthe illustrated embodiment, a plurality of self-alignment elements 408are formed at selected, predetermined locations within layer 400 andlayer 402. In the illustrated embodiment, self-alignment features 408comprise vertically disposed channels traversing, in some preferredembodiments, essentially completely through layers 400 and 402 such thatupon bringing layer 402 into conformal contact with layer 400 uppersurface 410 of layer 402 is in fluid communication with lower surface412 of layer 400 through vertically disposed channels formed by thealignment of the self-alignment elements contained within layers 400 and402 respectively. In other embodiments, one or more of the alignmentelements may not completely traverse the layer in which it is disposed,but may instead comprise an indentation, bump, or other feature withinor on the surface of the layer.

In order to effect proper self-alignment, it is important that layers400 and 402 be essentially identical in size and perimetric shape, whenviewed in the x-y plane along the negative z-axis direction asillustrated, such that the perimeter of layers 402 and 400 essentiallyidentically overlap when the layers are brought together into properlyaligned conformal contact. Optionally, in other embodiments, properself-alignment can also be effected if, instead of being essentiallyidentical in size and perimetric shape, one of the layers is much largerthan the other so that the meniscus of liquid formed around the edge ofthe smaller layer does not change appreciably in total surface area withsmall movements of the two layers with respect to each other.

Self-alignment elements 408, in preferred embodiments, are formed withinlayers 400 and 402, during the replica molding process for forming thelayers, by topological features provided within the mold mastersutilized for molding. Such topological features can be positioned andlocated within the mold master surface at selected, strategic positionswith respect to features within the mold master surface for formingchannels 404 and 406 through use of a CAD computer program, such asdescribed above for designing the overall layout of the topologicalfeatures for forming the various channels within the replica moldedlayer structures. Topological features forming self-alignment elements408 are positioned with respect to topological features forming channelstructures 404 and 406 so that when layer 400 and layer 402 aresuperimposed such that alignment holes 408 are precisely aligned withrespect to each other, channel features 404 and 406 are also orientedwith respect to each other in a desired registered alignment.

FIG. 6 b and FIG. 6 c illustrate the manner by which alignment holes 408interact with a fluid layer 412 disposed between layers 400 and 402 toeffect self-alignment. When self-alignment holes 408 and features 404and 406 are properly aligned with respect to each other, as shown inFIG. 6 b, the layers are arranged in an equilibrium position in whichthe interfacial area 414 between fluid layer 412 and the surroundinggaseous environment is minimized and there are no net capillary forces,due to the surface tension of fluid layer 412, tending to change theposition of layer 400 or layer 402 with respect to each other.

By contrast, when features 404, 406, and self-alignment holes 408 aremisaligned with respect to each other, as illustrated in FIG. 6 c, theinterfacial area 414 between fluid layer 412 and the surrounding gaseousatmosphere is increased with respect to the interfacial surface areawhen the system is in its equilibrium position as shown in FIG. 6 babove, and there will be a net resulting capillary force in thedirection shown by arrow 416, due to surface tension effects of fluidlayer 412, tending to bring the system into the equilibrium positionillustrated in FIG. 6 b.

In alternative embodiments, an essentially identical self-aligningeffect as illustrated in FIGS. 6 a-6 c can be achieved without the needfor forming self-alignment holes or features, such as 408, in the layerswhich are to be self-aligned with respect to each other. In suchalternative embodiments, the layers can be formed without self-alignmentholes, such as 408, but instead be formed or trimmed to have perimetershapes, which are essentially identical to each other, so that thelayers when stacked upon one another with a fluid layer therebetween, asillustrated in FIGS. 6 b and 6 c, will have a minimum free energyequilibrium position defined by an essentially precise and exact overlayof the essentially identical perimetric shapes of the two layers. Thefeatures comprising channels within the layers are, in such embodiments,strategically positioned with respect to the peripheral border of thelayers, so that, when the layers are aligned in the above-describedminimum energy, no net capillary force equilibrium position, theperimeters of the layers are precisely superimposed upon each other andthe features comprising the channels within the layers are alsosimilarly aligned with respect to each other in a desired registration.FIG. 6 d illustrates one contemplated embodiment of a perimetric shapefor enabling the above-described self-alignment of various layers of thestructure without the need for alignment holes.

The above-described self-alignment techniques are able to self-align astack of as many individual layers as is desired, essentiallysimultaneously and in parallel. The self-alignment technique describedherein is also capable of self-aligning elements with respect to eachother within a margin of error of approximately +/−10 μm or less,providing sufficient alignment precision for most of the channel sizesand configurations contemplated for the structures provided according tothe invention (e.g., channel structures having a cross-sectionaldimension ranging from about 20 μm to about 500 μm). The alignmentprecision obtainable by the above-described self-alignment technique istypically comparable or better than that obtainable via manual alignmenttechniques utilizing a stereomicroscope and conventionalmicromanipulation equipment.

The above-described self-alignment techniques are especially well suitedfor embodiments involving alignment of oxidized PDMS layers utilizingthe above-described alignment/sealing method using a non-reactive liquiddisposed between and able to wet the oxidized PDMS layers. However,those of ordinary skill in the art will readily realized that theabove-described self-alignment technique can be utilized for aligninglayers comprised of essentially any of the suitable materials forforming the microfluidic system discussed above and can be utilized forself-aligning layers that are not reactive with respect to each otherand do not become essentially irreversibly sealed to each other uponcontact but, instead, are simply aligned in conformal, non-sealingcontact with each other. Those of ordinary skill in the art can readilyselect appropriate liquids having desired surface-wetting properties(for use in the self-aligning technique when utilizing the technique toself align surfaces comprised of materials other than oxidized PDMS)using no more than known, published surface wetting properties forvarious liquids on various surfaces or routine screening tests notrequiring undue experimentation. In addition, while the above-describedself-alignment technique has been exemplified in the context of aligningtwo replica molded layers of the overall microfluidic structure withrespect to each other. In other embodiments, the technique can beutilized to-align a replica molded layer comprising one or more levelsof the microfluidic structure to the surface of a substrate, for examplea silicon microchip, or the like. Utilization of the self-aligningmethod for aligning a layer of the microfluidic network to a substratesurface, for example a surface of a silicon microchip, may be importantfor applications where the microfluidic network is utilized as anon-chip sensor, detector, analyzer, etc.

FIG. 7 illustrates an alternative embodiment for fabricating amicrofluidic network structure according to the invention. Unlike themethod previously described in the context of FIGS. 5 a-5 c, thefabrication method described in FIG. 7 involves the formation, byreplica molding, alignment, and assembly of only two, as opposed tothree, discrete layers forming the three levels of the overallmicrofluidic network structure.

As described above in the context of FIGS. 5 a-5 c, the method outlinedin FIG. 7 can potentially utilize a wide variety of hardenable liquidsfor forming the replica molded components of the microfluidic networkstructure. Such hardenable liquids were described previously in thecontext of FIGS. 5 a-5 c. As previously, in preferred embodiments, thereplica molded structure is formed of a polymeric material, morepreferably an elastomeric material, and most preferably a transparentelastomeric material. In a particularly preferred embodiment illustratedand exemplified in FIG. 7, the replica molded structures are formed ofPDMS.

In Step 1 of the method illustrated in FIG. 7, a mold master 500 havinga surface 502 including a series of topological features 504 thereonprotruding from the surface in positive relief is formed in a manneressentially equivalent to that described for forming mold master 300 ofFIG. 5 a. Topological features 504 are shaped, sized, and laid out onsurface 502 in a pattern predetermined to form a desired arrangement ofchannels disposed in the upper, third level of the overall microfluidicnetwork structure. Mold master 502 is then placed in the bottom of apetri dish or other container having a depth exceeding the height of theupper surfaces of topological features 504 on surface 502.

In Step 2, a hardenable liquid is added to the container containingmaster 500 in an amount sufficient to completely cover and submergetopological features 504. As discussed in FIG. 5 a above, surface 502 ofmold master 500, in preferred embodiments, is treated with a releaseagent, for example a silanizing agent, to permit release of the replicamolded structure from the surface without undue damage or distortion ofthe replica molded structure. Also in Step 2, as described above in thecontext of FIGS. 5 a-5 c, the hardenable liquid, for example a PDMSprepolymer solution, is cured and solidified to form a solid moldedreplica 510 of surface 502 of mold master 500. Molded replica 510 isremoved from surface 502 after curing as illustrated in Step 2. In theillustrated embodiment, molded replica 510 comprises a PDMS slab whichcan, as illustrated, be trimmed to a desired overall size and perimetricshape. Molded replica 510 includes therein, but not completely extendingtherethrough, a series of indentations 512 in lower surface 514corresponding to topological features 504 of mold master 500.Indentations 512 form channels disposed within the third, upper level ofthe overall microfluidic network to be fabricated.

Steps 3 and 4 of the method illustrated in FIG. 7 comprise the formationof a replica molded membrane layer including therein both channelsdisposed in the first, lower level of the overall microfluidic networkstructure and connecting channels of the third, intermediate level ofthe overall microfluidic network structure forming fluidic connectionsbetween the channels disposed in the first, lower level and the second,upper level of the structure. The molded replica membrane layer, havingtwo levels of features formed therein, is formed by a membrane sandwichfabrication method (Steps 3 and 4) similar to the method previouslydescribed in the context of FIGS. 5 a-5 c, except that mold master 520includes a surface 522 having formed thereon a plurality of topologicalfeatures 524 in positive relief protruding from surface 522, thatinclude features, for example feature 526, that are two-leveltopological features, which are characterized by a first portion 528having a first height with respect to a region of surface 522 adjacentto feature 526 and a second portion 530, which is integrally connectedto the first portion, having a second height with respect to surface 522adjacent feature 526, which second height is greater than the height offirst portion 528.

The term “integrally connected,” as used herein in the context ofdescribing two-level topological features of mold masters, refers tosuch features having at least a first portion and a second portion, thesecond portion having a height or depth with respect to the surface ofthe mold master adjacent the feature different from the first portion,wherein the first and second portion comprise two different regions of acontinuous structure or comprise two discrete structures each having atleast one surface in direct contact with at least one surface of theother. By providing such two-level topological features on mold master520, the illustrated method allows simultaneous formation and alignmentof channels disposed within two levels of the overall microfluidicnetwork structure. Thus, by forming two levels of the overall structurewithin a single layer in a single replica molding step, the presentmethod eliminates the need to align two discrete layers comprising thefirst, lower level of the structure and the third, intermediated levelof the structure with respect to each other after formation of themolded replica layers. Thus, as described below, the present methodrequires only a single alignment step for assembling the molded replicalayers into the overall microfluidic network structure.

A variety of photolithography and micromachining methods known to thoseof ordinary skill in the art, which are capable of forming features on asurface having multiple heights or depths with respect to the surface,can potentially be utilized in the context of the present invention forforming the two-level topological features 526 of mold master 520. Aparticularly preferred embodiment for forming mold master 520 involvesan inventive method for forming two-level topological features inphotoresist, and is described in more detail below in the context ofFIG. 8.

After formation of mold master 520, a layer of hardenable liquid, forexample PDMS, is placed upon surface 522 of mold master 520 and coveredwith an upper mold master 540, having a lower surface 542 that isessentially flat and featureless, so that surface 542 is in conformalcontact with the uppermost surfaces of the two-level topologicalfeatures 526 on surface 522 of mold master 520. As previously describedin the context of FIGS. 5 a-5 c, mold master 540 can comprise a varietyof materials including, for example, an elastomeric polymer slab, forexample formed of PDMS, a polymeric sheet, a flat silicon wafer, etc. Inpreferred embodiments, as previously discussed, it is desirable that theinterfacial adhesion strength between surface 522 of mold master 520 andthe hardened molded replica differ from the interfacial surface adhesionbetween surface 542 of mold master 540 and the hardened liquidcomprising the molded replica. In the illustrated embodiment, surface522 of mold master 520 comprises a silanized polymeric negativephotoresist layer and mold master 540 comprises a Teflon™ (PTFE) sheet.

In Step 4, pressure is uniformly applied to surface 544 of upper moldmaster 540 and surface 546 of lower mold master 520 to enable the uppersurfaces 548 of topological features 526 to make sealing contact withsurface 542 of mold master 540 during the hardening and curing processforming the replica molded membrane layer. In Step 4, the hardenableliquid, for example PDMS prepolymer, is cured to form a two-levelreplica molded membrane 550. Two-level replica molded membrane 550includes a plurality of first channels 552, disposed within a lowersurface 554 of the membrane, comprising channels disposed within thefirst level of the overall microfluidic network structure, and alsoincludes vertically oriented connecting channels 554 that completelypenetrate the thickness of the membrane and interconnect lower surface554 and upper surface 556 of the membrane, forming the connectingchannels disposed within the third, intermediate level of the overallmicrofluidic network structure. Channels 552 comprise replica moldedfeatures corresponding to first portions 528 of topological features 526of mold master 520 and connecting channels 555 comprise replica moldedfeatures corresponding to second portions 530 of two-level topologicalfeatures 526 of mold master 520.

In the illustrated embodiment, the PDMS membrane comprising moldedreplica layer 550 is separated from the mold masters by first peelingPTFE sheet 540 from the upper surface 556 of the membrane andsubsequently peeling the membrane from upper surface 522 of mold master520. In other embodiments, molded replica 550 can remain in contact withupper surface 522 of mold master 520 during the subsequent, and belowdescribed, aligning and sealing steps, in order to support membrane 550and prevent distortion or destruction of the molded features therein. Itshould be understood, that for more complex structures, additionalreplica molded membranes such as 550 can be stacked upon each other inthe assembly of the microfluidic network structure to yield structureshaving more than three levels of interconnected microfluidic channels.

In the final step of fabrication, Step 5, replica molded slab 510 andreplica molded membrane 550 are aligned with respect to each other toyield the desired microfluidic network structure, brought into conformalcontact with each other, and optionally sealed together by methodspreviously described above in the context of FIGS. 5 a-5 c to yield thefinal microfluidic network structure 560. As previously described, thestructure 560 can include inlet conduits 562 and outlet conduits 564 foreach of the non-interconnected fluid flow paths disposed within thestructure, or other interconnections between the flow paths within thestructure and the external environment as required or desired for aparticular application. In the illustrated embodiment, microfluidicnetwork structure 560 includes three non-fluidically interconnectedfluid flow paths therein. The first flow path 561 has an inlet andoutlet in the foreground and is shaded light gray; the second flow path563 has an inlet and outlet that are centrally disposed shaded in black;and the third flow path 565 has an inlet and outlet in the backgroundand is shaded dark gray.

In addition, lowermost surface 554 of structure 560 includes therein apattern indentations corresponding to the channels of the first, lowerlevel of the microfluidic network structure formed within the bottomsurface 554 of the replica molded membrane 550. Thus, microfluidicnetwork structure 560 is useful for embodiments wherein the microfluidicnetwork structure is utilized as a surface patterning stamp fordepositing materials onto a material surface in a pattern correspondingto the channels disposed within surface 554, or otherwise creating apatterned surface with a pattern corresponding to the pattern of thechannels disposed within surface 554. In alternative embodiments,surface 554 can be placed in conformal contact with, and optionallysealed to, a solid PDMS slab, or other substrate or surface, to form anenclosed microfluidic network structure, as described previously in thecontext of FIGS. 5 a-5 c.

FIG. 8 illustrates a preferred method for preparing mold masters thathave a surface including thereon one or more two-level topologicalfeatures. While the illustrated method is useful for forming two-leveltopological features in layers of either negative or positivephotoresist materials, in the embodiment illustrated, a negativephotoresist material (e.g., SU8-50) is utilized as an example. Inaddition, while, in the illustrated embodiment, two-level topologicalfeatures comprising positive, high-relief features protruding from thesurface of the mold master are fabrinated, it should be understood thatthe method is also well suited to produce two-level topological featurescomprising negative, low-relief features characterized by indentations,grooves, or channels within the surface of the mold master. Anyvariations in the below described technique for producing two-levelpositive, high-relief features in negative photoresist that are requiredin order to produce two-level features in positive photoresist and/or toproduce two-level features comprising negative, low-relief featuresinvolve only simple extensions of the below-described method that wouldbe apparent to those of ordinary skill in the art.

In Step 1 of the method illustrated in FIG. 8, a silicon wafer 600, orother suitable substrate, is coated with a layer of photoresist 602, bya conventional spin-coating technique or other suitable coatingtechnique known to those of ordinary skill in the art. Layer 602 isspin-coated to a depth corresponding to the desired depth of the deepestfeature to be formed on the first level of the mold master (e.g. a depthcorresponding to the deepest channel to be disposed in the level of themicro fluidic channel structure to be replica molded by the first Ievelof the mold master. The thickness of layer 602 will typically range fromabout 20 μm to about 500 μm, and can, in some embodiments, be as thickas about 1 mm.

In Step 2, the photoresist is “soft baked” by being exposed to anelevated temperature for a short period of time to drive off solventused in the spin-coating process For example, for SU8-50 negativephotoresist, the coated substrate is exposed to a temperature of about95-105° C. for a period of several-minutes. In Step 3, a-first photomask604 including thereon a pattern 606 corresponding to features 626 of thefirst level of the mold master is placed in contact with negativephotoresist layer 602. As would be apparent to those of ordinary skillin the art, a wide variety of photomasks can be utilized according tothe present inventive method; however, in a preferred embodimentillustrated, photomask 604 comprises a high resolution transparency filmhaving a pattern printed thereon. Designs for the channel system printedupon the high resolution transparency are preferably generated with aCAD computer program. In the illustrated embodiment, a high-resolution(e.g., 3000-5000 dpi) transparency, which acts as photomask 604, isproduced by a commercial printer from the CAD program design file. Inthe illustrated embodiment, essentially the entirety of photomask 604 isrendered opaque to the radiation used to expose the photoresist by alayer of toner, and the fluidic channel-forming features to be formed onthe surface of negative photoresist 602 correspond to transparentregions 606 of the photomask surface.

In addition to regions 606 corresponding to features in the mold masterfor formning fluidic channels within the molded replica structure formedwith the mold master, photomask 604 also includes peripheral transparentregions 608, which correspond to topological features for formingalignment tracks useful for aligning the mold masters with respect toeach other in certain methods for forming microfluidic structures asdescribed in more detail below in FIGS. 9 a and 9 b.

In Step 4, upper surface 603 of photoresist layer 602 is exposed toradiation, for example ultraviolet (UJV) radiation of a frequency andintensity selected to cross-link exposed areas of the negativephotoresist, through the transparent regions of the printed pattern ofphotomask 604. In Step 5, after exposure to cross-linking radiation, thefirst photomask 604 is removed from the surface, the photoresist ishard-baked (e.g. at about 95-105° C. for several minutes) and a secondlayer of photoresist is spin-coated on top of surface 603 of photoresist602. The second layer of photoresist is spin-coated to a thicknesssufficient for forming features in the mold master corresponding to theconnecting channels disposed within the third, intermediate level of thereplica molded microfluidic network structure formed with the moldmaster. Typically, the thickness of the second level of photoresist willrange from about 20 μm to about 1 mm. Wafer 600, now containing a first,exposed layer of photoresist and a second layer of unexposed photoresistcan then be subject to another soft-baked procedure to drive off solventfrom the unexposed layer of photoresist, similarly as described in Step2 above.

As illustrated in Step 5 of FIG. 8, regions of the first layer ofphotoresist that were exposed to the radiation (e.g., regions 610 and612) typically exhibit a change in the degree of transparency and/orrefractive index of the photoresist, thus rendering them visible throughthe upper layer of newly spin-cast, unexposed photoresist. Thisvisibility allows a second photomask to be easily aligned with respectto the first exposed pattern by using a standard photomask aligner. Inother embodiments, especially where the exposed pattern may not bevisually apparent, visible alignment features or elements can beincluded on the surface of wafer 600 to enable alignment of the secondphotomask to achieve a desired two-level pattern, as would be apparentto those of ordinary skill in the art.

In Step 6, a second photomask 614 including thereon printed patterns616, corresponding the second level portions of the two-leveltopological features of the mold master, which form the connectingchannels in the intermediate level of the replica molded microfluidicnetwork structure formed with the mold master, and 618, corresponding toa second level of the optional alignment tracks. It should beunderstood, that while, in the illustrated embodiment, features 606corresponding to topological features for forming channels disposed inthe first level of the microfluidic network structure comprise linearfeatures, in other embodiments, features 606 can be non-linear, thusforming curved topological features resulting in non-linear, curvedchannels within the first level of the microfluidic structure.Similarly, any of the previously described structures and methods forforming channels disposed within a particular level of microfluidicnetwork structure can include channels that are non-linear and curvedwithin the plane or curved surface defining the level of themicrofluidic network structure in which the channels are disposed inaddition to, or instead of, the straight channels previouslyillustrated.

Printed pattern 616, creating topological features for forming channelswithin the microfluidic network structure can also, in some embodiments,include features parallel and contiguous with regions 610 formed withinthe first layer of photoresist and corresponding to printed pattern 606,such that some of the topological features produced on the surface ofthe mold master by the illustrated method include features that formchannels having a longitudinal axis parallel to the first level of thereplica molded microfluidic network structure formed with the moldmaster, and which have an overall depth within the replica moldedmicrofluidic network structure formed with the mold master, which isequal to the combined depth of the first level and the third,intermediate level of the structure (i.e., for forming replica moldedmicrofluidic network structures having deep channels that are disposedwithin both the first level and the third, intermediate level of themicrofluidic network structure).

Photomask 614 is aligned in Step 6 with respect to exposed pattern 610and the second, unexposed layer of photoresist is exposed, in Step 7, tothe cross-linking radiation through photomask 614. Following exposure,mask 614 is removed from the top layer of photoresist, and thephotoresist is hard-baked as described above. If desired, theabove-mentioned steps can be repeated with additional layers ofphotoresist and additional photomasks to produce more than two levels oftopological features on the surface of wafer 600. After the desirednumber of layers of photoresist have been coated onto wafer 600 andexposed to cross-linking radiation as described above, the reliefpattern is developed in Step 8 by exposing the photoresist to adeveloping agent that dissolves and removes non-crosslinked photoresistmaterial leaving behind a mold master 620 having a surface 622 includingthereon a pattern of two-level high relief features 624 having a firstportion 626 with a first height above surface 622 and a second portion628 having a second height above surface 622, which is greater thanheight 626. First portion 626 of the topological features forms thechannels disposed within the first level of the replica moldedmicrofluidic network structure formed with mold master 620, and secondportion 628 of the topological features forms the connecting channelstraversing the third, intermediate level of a microfluidic networkstructure replica molded using mold master 620.

Also formed on surface 622 of mold master 620 by the above-outlinedprocess are alignment tracks 630 having a height corresponding to theheight of the second portion 628 of topological features 624. While, inthe illustrated embodiment, the second layer of photoresist wasspin-coated onto a first layer of exposed photoresist before developingthe first layer, in an alternative embodiment, the first layer ofphotoresist can be developed before spin-coating the second layer ofphotoresist if desired. Solvents useful for developing the unexposedportions of the photoresist are selected based on the particularphotoresist material employed. Such developing agents are well known tothose of ordinary skill in the art and are typically specified by thecommercial manufacturers of many of the photoresists useful forperforming the methods of the invention. For example, for theillustrated embodiment utilizing SU8-50 negative photoresist,uncross-linked photoresist can be removed during development by exposingthe photoresist to propylene glycol methyl ether acetate. Two-level moldmaster 620, subsequent to formation as described above, is preferablycoated with a release agent, for example by silanizing the surface, inorder to facilitate removal of a molded replica from the surface of themold master.

FIGS. 9 a and 9 b illustrate the steps of a third embodiment of themethod according to the invention for fabricating a three-dimensionalmicrofluidic network structure. The method illustrated in FIGS. 9 a and9 b comprises a membrane sandwich technique similar to that previouslydescribed in Steps 3 and 4 of the method illustrated in FIG. 7, exceptthat instead of forming a replica molded membrane layer between a bottommaster including two-level topological features and a top mold masterhaving an essentially flat, planar surface, as was illustrated in themethod of FIG. 7, in the method according to FIGS. 9 a and 9 b, areplica molded membrane layer is formed between two mold masters, bothincluding topological features thereon and at least one including atleast one two-level topological feature thereon, thus yielding a replicamolded membrane including therein a microfluidic network structurecontaining all three of the above-discussed levels. In some embodiments,both the upper and lower mold masters utilized for forming thethree-level replica molded membrane layer according to the embodiment ofFIGS. 9 a and 9 b can comprise mold masters, for example similar to moldmasters 500 and 520 shown in FIG. 7. However, as previously discussed,it is desirable for at least one of the mold masters to be formed of anelastomeric material to improve sealing contact between portions of thesurfaces of the mold masters that are in contact during the replicamolding process so as to prevent undesirable leakage of hardenableliquid into such regions of contact. Therefore, in preferredembodiments, the upper mold master and/or lower mold master are formedfrom an elastomeric material having a surface with topological featuresthereon.

In some particularly preferred embodiments, elastomeric mold masters areformed using a replica molding procedure, similar to that used to formthe various layers of the microfluidic structure, to form topologicalfeatures on the elastomeric mold master that are formed during replicamolding from topological features on a pre-master prepared byphotolithography or micromachining. The method illustrated in FIGS. 9 aand 9 b correspond to such a preferred embodiment. In the illustratedembodiment, the top mold master, as well as the replica molded membranelayer, are formed from an elastomeric material comprising PDMS. Asreferred to and discussed extensively above, PDMS, while being preferredfor forming many of the structures and mold masters according to theinvention, comprises only one example of a material formable from ahardenable liquid useful for forming the mold masters and microfluidicnetworks according to the invention. A wide variety of alternativematerials and hardenable liquids have been previously discussed in thecontext of the methods illustrated in FIGS. 5 and 7, and such materials,or other materials apparent to those of ordinary skill in the art, canbe substituted for PDMS in the method illustrated in FIGS. 9 a and 9 bbelow.

FIG. 9 a illustrates one preferred method for forming an elastomeric topmold master for use in forming a three-level replica molded membranelayer. In Step 1, a pre-master mold is fabricated by forming topologicalfeatures on a surface of a substrate 700, for example as previouslyillustrated in the context of FIG. 8. Since, in the illustratedembodiment, it is desired that the topological features formed in thereplica molded top mold master comprise positive, high-level relieffeatures protruding from the surface of the mold master, the topologicalfeatures formed on surface 702 of substrate 700 comprise negative,low-level relief features characterized by grooves or channels 704, 706seen more clearly in the cross-sectional view. In the illustratedembodiment, pre-master mold 700 is fabricated using a twolevelphotolithography technique similar to that described in FIG. 8.Topological features 706 have a greater depth than topological features704 and essentially traverse the entire thickness of photoresist layer708. In the illustrated embodiment, topological features 706 correspondto and form topological features in the replica molded elastomeric moldmaster which are alignment tracks, whose function is explained in moredetail below. Topological features 704 correspond to and formtopological features in the replica molded mold master which areresponsible for forming channels ultimately disposed in the second,upper level of the replica molded three-level membrane layer. It shouldbe understood that in alternative embodiments, one or more oftopological features 704 can comprise two-level topological featureshaving a first portion with a first depth with respect to surface 702and a second portion with a second, greater depth (e.g. corresponding tothe depth of topological features 706) with respect to surface 702. Forsuch embodiments, a replica molded top mold master would includetwo-level topological features in positive relief for forming channelsdisposed in the second, upper level of the replica molded membrane aswell as connecting channels traversing the membrane. In suchembodiments, the lower mold master can include channel-formingtopological features having a single, uniform height or can includechannel-forming topological features that are also two-level topologicalfeatures.

In Step 2, pre-master mold 700 is placed into the bottom of container712. The container is then filled with a hardenable liquid, such as PDMSprepolymer, to a level at least covering upper surface 702 of pre-mastermold 700. Subsequently, the hardenable liquid is cured or solidified, aspreviously discussed, and, in Step 3, is removed from the pre-mastermold, optionally trimmed, and treated with a release agent, for exampleby silanization or oxidation followed by silanization. The resultingstructure 720 comprises a replica molded mold master including a surface722 having disposed thereon topological features 724 at a first heightwith respect to surface 722 and corresponding to topological features704 of pre-master 700, and topological features 726 having a second,greater height with respect to surface 722 and corresponding totopological features 706 on pre-master 700. Topological features 724comprise channel-forming features and topological features 726 comprisealignment tracks.

FIG. 9 b illustrates steps for forming the replica molded three-levelmembrane layer with the upper mold master 720 produced according to thesteps outlined in FIG. 9 a above and a lower mold master 620 producedaccording to the method outlined previously in FIG. 8. In Step 4, aquantity of hardenable liquid 310, for example PDMS prepolymer, isplaced in contact with upper surface 622 of lower mold master 620 in anamount sufficient to form a layer having a thickness at least equal tothe height of topological features 628 and 630. Upper mold master 720 isthen brought into contact with lower mold master 620 in Step 5 and ismanually manipulated until topological features 726 comprising alignmenttracks in the upper mold master mate and interdigitate with topologicalfeatures 630 comprising alignment tracks in the lower mold master. Uponmating and interdigitating of alignment tracks 726 and 630, thealignment and relative position of channel-forming topological features724 of the upper mold master and channel-forming topological features624 of the lower mold master is such that they are properly positionedand aligned with respect to each other to form the desiredthree-dimensional microfluidic network channel structure within thereplica molded membrane layer. The interface between the upper moldmaster 720 and lower mold master 620 during the replica molding processin Step 5 is seen more clearly in the cross-sectional view. Thecross-sectional view illustrates that, upon proper alignment, alignmenttracks 726 of upper mold master 720 mate and interdigitate withalignment tracks 630 in lower mold master 620. In addition, thecross-sectional view also clearly illustrates the conformal, sealingcontact made between channel-forming feature 725 in upper mold master720 and the upper surface of second portions 628 of the topologicalfeatures on the surface of the lower mold master.

In Step 6, hardenable liquid 310, for example PDMS prepolymer, is cured,as previously described and upper mold master 720 is peeled away fromlower mold master 620. In the illustrated embodiment, where upper moldmaster 720 comprises silanized PDMS, lower mold master 620 has an uppersurface 622 comprising polymeric photoresist and hardenable liquid 310comprises PDMS prepolymer, the replica molded PDMS membrane layer 730formed upon curing will adhere more strongly to surface 722 of uppermold master 720 than to surface 622 of lower mold master 620 and, uponpeeling away of upper mold master 720, will remain adhered to andsupported by upper mold master 720, thus preventing damage to themembrane.

Replica molded membrane layer 730 includes therein channels 732 disposedwithin lower surface 734 of membrane 730, formed by first portion 626 oftopological features 624 of lower mold master 620; upper channels 736disposed within upper surface 738 of the membrane, formed by topologicalfeatures 724 of the upper mold master; and connecting channels 740traversing the membrane and interconnecting surface 734 and surface 738,which interconnecting channels are formed by second portions 628 oftwo-level topological features 624 of lower mold master 620. Thus, inthe presently described method, a single replica molded layer is formedthat includes therein all three levels required to form athree-dimensional microfluidic network structures according to theinvention. In addition, because of the provision of alignment tracks 726and 630, the entire three-dimensional network structure was formedwithout the need for performing an alignment of features or channelsrequiring the use of a microscope or micromanipulator. Because thepresent method does not require visual alignment of features orchannels, it can be especially useful for forming microfluidic membranestructures from materials that are opaque to visible light.

When, as illustrated, the three-level membrane is formed by utilizingone mold master formed via a photolithographic or micromachiningtechnique (e.g. mold master 620) together with an elastomeric moldmaster (e.g. 720), which is formed by replica molding a pre-master moldformed via a photolithographic or micromachining technique (e.g.pre-master 700), if the hardenable liquid utilized to form the replicamolded mold master (e.g. as illustrated in Step 2 of FIG. 9 a) has atendency to shrink during hardening, this shrinkage should be taken intoaccount when sizing and positioning the topological features of thepre-master, so that topological features of the replica molded moldmaster will properly match those of the other mold master to yield thedesired alignment of channels. For example, when PDMS is used to formone mold master, it has been found that the size and relative spacing ofthe features in the pre-master should be increased by about 0.66% overthat desired for the final PDMS mold master in order to account forshrinkage of the mold master during curing of the pre-polymer.

Replica molded polymeric membrane 730 can be removed from upper moldmaster 720 and can be utilized as a stand-alone structure or can bestacked with other such structures to form more complex networks.Optionally, and as shown in Step 7, before removal from upper moldmaster 720, lower surface 734 of membrane 730 can be brought intoconformal contact with a lower substrate layer 750, for example, a flatpiece of PDMS, silicon wafer, microchip, or other substrate, and canoptionally be sealed thereto as previously described. Substrate layers,instead of having flat smooth surfaces as illustrated, can, in otherembodiments, include topological features thereon that are matable withtopological features on the surface of the replica molded membrane, forexample, alignment tracks 739, so that, upon interdigitation of thematable topological features on the substrate layer and one or moretopological features on the surface of the replica molded membrane, themembrane is aligned and oriented in a desired configuration with respectto the substrate.

After contacting the membrane with the substrate layer and, optionally,essentially irreversibly sealing the membrane to the substrate layer,upper mold master 720 can then be removed from upper surface 738 ofmembrane 730 as illustrated in step 8. The resulting microfluidicnetwork structure 760 can be utilized as shown or after trimming awaythe regions of the membrane including alignment tracks 739. Structure760 is useful, for example, as a microfluidic membrane stamp forpatterning a material surface, the stamping surface comprising uppersurface 738 of membrane 730, which has channels 736 disposed therein.Structure 760 is also useful for embodiments wherein the microfluidicnetwork structure is utilized as a mold in which to formthree-dimensional networks of materials having a structure correspondingto the channel structure in membrane 730, as described in more detailbelow.

For embodiments where it is desired to provide an enclosed series ofmicrofluidic channels, upper surface 738 of membrane 730 is subsequentlyplaced in conformal contact with and, optionally sealed to, an uppersubstrate layer 770. Upper substrate layer 770 can comprise a slab ofPDMS or other substrate layer desirable for a particular application, aspreviously discussed. Also, as previously discussed, inlet and outletconduits can be formed within either or both of substrate layers 770 and750 in order to interconnect the fluid flow paths of the microfluidicchannel structure to the external environment.

FIG. 9 c illustrates a modification of the embodiment for fabricatingthe three-dimensional microfluidic structure, as illustrated in FIGS. 9a and 9 b. In the modification illustrated in FIG. 9C, the upper andlower mold masters utilized for forming the three-level replica moldedmembrane layer each include two-level topological features thereon forforming the connecting channels traversing the replica-molded membrane.

The two-level features of the upper and lower mold masters that form theconnecting channels through the membrane are configured to havecomplementary, mateable shapes, such that when the mold masters areplaced together during the replica molding step (e.g., step 5 asillustrated in FIG. 9 b), the mateable, channel-forming topologicalfeatures on the upper and lower mold masters will mate/interdigitatewith each other, for example, as shown in FIG. 9 c(iv). Providing suchmateable, connecting channel-forming features can reduce any tendency ofthe hardenable liquid for forming the replica molded membrane to beincompletely excluded from the regions forming the connecting channelsduring the molding process, which incomplete exclusion can lead to theformation of an undesirable, thin layer of hardened polymer occludingthe connecting channels after molding. In the modified embodimentillustrated, wherein the two-level topological features of the upper andlower mold master that form the connecting channels are configured withshapes that are mateable with each other, the hardenable liquid can bemore effectively and thoroughly excluded from the region molding theconnecting channels, thus effectively eliminating any tendency to form athin film of hardened material occluding the connecting channels uponformation of the membrane.

In addition, the mateable, connecting channel-forming two-leveltopological features can also serve a purpose similar to that of thealignment tracks discussed above. Namely, upon mating or interdigitationof the mateable, connecting channel-forming features of the upper andlower mold master, the alignment and relative position of the variousother channel-forming topological features of the upper and lower moldmaster will be properly positioned and aligned with respect to eachother to form the desired three-dimensional microfluidic network channelstructure within the replica molded membrane layer. Also, relativemotion between the upper and lower mold masters, leading tomisalignment, during the replica molding step can be reduced oreliminated. Accordingly, although the alignment track-forming featuresare illustrated in the modified embodiment shown in FIG. 9 c, becausethe mateably-shaped connecting channel-forming topological features ofthe upper and lower mold master can perform essentially the samefunction and fulfil essentially the same purpose, in some embodimentsutilizing the modified mold masters, the alignment track-formingfeatures could be eliminated.

FIG. 9 c(i) and (ii) illustrate a modified pre-master mold 781 forforming the replica molded upper mold master 782 that includes thetwo-level connecting channel-forming topological features configured tomate/interdigitate with complementary connecting channel-formingfeatures in the lower mold master. Pre-master mold 781 can be fabricatedby forming topological features on surface 702, for example aspreviously illustrated in the context of FIGS. 8 and 9 a. As previouslydescribed in the context of FIG. 9 a, since it is desired that thetopological features formed in the replica molded top mold mastercomprised positive, high-level relief features protruding from thesurface of the mold master, the topological features formed on surface702 comprise negative, low-level relief features characterized bygrooves or channels 704, 706, and 783. In the illustrated embodiment,pre-master mold 781 is fabricated using a two-level formingphotolithography technique similar to that described above in FIG. 8.

One-level channel-forming feature 704, and two-level alignment trackforming feature 706 are essentially identical to those previouslydescribed in the context of FIG. 9 a. In contrast to the embodimentillustrated previously in FIG. 9 a, however, pre-master mold 781includes a topological feature 784 corresponding to and forming atopological feature in the replica molded mold master, which isresponsible for forming a channel ultimately disposed in the second,upper-level of the replica molded three-level membrane layer, whichfeature 784 includes, and is bounded by, topological features 783, whichare configured to form connecting channel-forming features in thereplica molded upper mold master that will have a shape that is mateableto complementary connecting channel-forming features in the lower moldmaster. Topological feature 783, shown in cross-section, comprises anouter ring 785 in two-level negative relief surrounding a central post786, the ring and post together forming a “donut”-shaped two-levelannulus.

FIG. 9 c(ii) illustrates the resulting upper mold master formed byreplica molding pre-master 781, as discussed previously in the contextof FIG. 9 a, Step 2. The resulting structure 782 comprises a replicamolded mold master including a surface 722 having disposed thereontopological features 724 and 787 at a first height 791 with respect tosurface 722 corresponding to topological features 704 and 784,respectively, of pre-master 781, and topological features 726 having asecond, greater height 794 with respect to surface 722 and correspondingto topological features 706 on pre-master 781. Upper mold master 782also includes topological features 788, corresponding to topologicalfeatures 783 of pre-master 781, features 788 including a central holeregion 790, in which the molded material comprising the mold masterextends to a position at the first height 791 with respect to surface722, and an outer peripheral ring 789 having a second, greater height794 with respect to surface 722. Topological features 788 comprisetwo-level, connecting channel-forming features, having a shape that ismateable to corresponding features on the lower mold master.

The lower mold master 792, illustrated in FIG. 9 c(iii) is substantiallysimilar to lower mold master 620 illustrated and discussed previously inthe context of FIGS. 8 and 9 b, however, the second portions (e.g.,portions 628 as illustrated in FIG. 9 b) of two-level topologicalfeatures 626 of FIG. 9 b, which are now called out by figure label 793,are somewhat smaller in diameter than those illustrated in FIG. 9 b, andare sized and positioned to mate and interdigitate with holes 790 ofinterconnecting channel-forming topological features 788 of upper moldmaster 782, when the mold masters are brought together and alligned forforming the three-level microfluidic membrane as illustrated in FIG. 9c(iv).

It should be understood that while, in the illustrated embodiment, theshape of the matable connecting channel-forming topological features ofthe upper mold master comprises a circular, donut-shape annulus, andthat of the lower mold master connecting channel-forming topologicalfeatures comprises a post, in other embodiments, this configurationcould be reversed such that the annulus-shaped features are present onthe lower mold master and the posts are present on the upper moldmaster. In addition, in other embodiments, upper mold master 782, asdiscussed previously, need not be a replica molded elastomericstructure, but instead could comprise a mold master formed inphotoresist, or other material, for example similar to lower mold master792, which could be formed by, for example a micro-machining techniqueor, more preferably, as previously discussed in the context of FIG. 8.

It should also be understood that while the mateable, interconnectingchannel-forming features illustrated in the present embodiment comprisea circular cylindrical post-annulus arrangement, in other embodiments,the interdigitating, mateable shapes of the interconnectingchannel-forming features of the upper and lower mold masters could beselected from an extremely wide variety of suitably mateable shapes. Forexample, instead of a circular post mating with an annulus having acircular centrally-disposed bore therein, a variety of alternativecylindrical shapes could instead be utilized, for example squares,triangles, rectangles, n-sided polygons, ovals, etc. Alternatively,mateable configurations other than a post-annulus configuration, asillustrated, could be employed. For example, one of the mold masterscould include interconnecting channel-forming features including a slotelement that is mateable with a corresponding groove element in theinterconnecting channel-forming features of the other mold master, or,alternatively, one mold master could provide interconnectingchannel-forming features including a half cylinder-shaped element withthe other mold master also providing interconnecting channel-formingfeatures including half cylinder-shaped elements, which halfcylinders-shaped elements of the first and second mold masters to matetogether to together form cylindrical interconnecting channel-formingfeatures. Those of ordinary skill in the art will readily envision awide variety of such mateable shapes and configurations suitable for usein the present context and providing substantially equivalent functionand performance as described above. Each of such alternativeconfigurations is deemed to be an equivalent structure falling withinthe scope of the present invention.

FIG. 10 illustrates a method for forming the five-level microfluidicnetwork structure, shown previously in FIG. 4 a, comprising a coilednetwork of interconnected channels forming a first fluid flow pathsurrounding a straight channel forming a second fluid flow path. Themethod in FIG. 10 is based upon the methods previously described inFIGS. 8, 9 a, and 9 b discussed above. In the method shown in FIG. 10,two separate molded replica membrane layers are formed, which aresubsequently aligned with respect to each other and sealed together toform the final, overall, five-level coiled network structure 220. Thefirst molded replica membrane layer 800 comprises three levels of theoverall structure and a second molded replica membrane layer 810comprises the remaining two levels of the overall microfluidic networkstructure. Molded replica layer 800 comprising three levels is formed bythe membrane sandwich method previously discussed in the context of FIG.9 b and utilizing a lower mold master 802 having formed thereon aplurality of two-level topological features 804 having first portions806, forming channels 807 disposed within the first, lowermost level ofthe overall microfluidic network structure, and second portions 808forming connecting channels traversing the level adjacent to andpositioned immediately above the lowermost level of the microfluidicnetwork structure upon replica molding.

Upper mold master 812 is preferably a replica molded elastomericmaterial (e.g. like mold master 720) and includes a bottom surface 814having a plurality of single-level topological features 816 protrudingtherefrom including a centrally disposed feature 818, forming thestraight channel 819 disposed on the second, upper level of membranelayer 800, and a plurality of features 820, aligned with second portions808 of topological features 804 of lower mold master 802, forming acontinuation of connecting channels 821 through the second, upper levelof replica molded layer 800 upon replica molding. Molded replica layer810, comprising the two-uppermost levels of the overall structure, isformed by the same membrane sandwich method utilizing lower mold master802 and an upper mold master 830, which comprises a flat slab ofpreferably elastomeric material. Two-level topological features 804,having first portions 806 and second portions 808, form a series ofchannels 832 disposed within lower surface 834 of molded replica layer810 and form connecting channels 833 traversing the thickness of moldedreplica layer 810, upon replica molding of layer 810.

In order to complete the assembly and form the overall coiledmicrofluidic network structure 220, molded replica layer 810 is rotated1800 in the direction of arrow 836, stacked on top of molded replicalayer 800, aligned so that the replica molded channels are registered toform the desired coiled channel network structure, brought intoconformal contact with, and optionally sealed to molded replica layer800. Optionally, surface 834 of molded replica layer 810 and/or surface809 of molded replica layer 800 can be brought into conformal contactwith, and optionally sealed to, a substrate layer (e.g., 838, 839) priorto or subsequent to stacking, aligning, and, optionally, sealing layers800 and 810 to each other. If desired, excess material comprising layers800 and 810 can be trimmed from the structure as illustrated in thefinal step of FIG. 10. The resulting structure 220 includes the coiled,two fluid flow path microfluidic network previously described in detailin the context of FIG. 4 a above.

In addition to being useful as fluid flow directing networks forapplications requiring fluid management in very small scale devices, forexample, in micro total analysis systems (μTAS), the microfluidicnetwork structures provided according to the invention are also usefulfor a variety of other uses. For example, microfluidic channel systemsfabricated according to the invention can be used to fabricate a varietyof microstructures having three-dimensional structures corresponding toa three-dimensional network of channels within a microfluidic networkstructure. Such microstructures can be formed by filling the channelnetwork of the microfluidic systems with a hardenable liquid,solidifying the hardenable liquid within the network channels, and,optionally, removing the surrounding microfluidic network structure toyield a free-standing microstructure comprised of the solidifiedhardenable liquid. The hardenable liquid utilized for formmicrostructures that are replica molded within the inventivemicrofluidic network systems can comprise essentially any of thehardenable liquids described above as being useful for forming themicrofluidic network structures themselves. The hardenable liquidschosen to form the replica molded microstructures should be chemicallycompatible with the microfluidic network structure and, for embodimentswhere it is desired to selectively remove a surrounding microfluidicnetwork structure, should be resistant, once hardened, to whatevertreatment is required to dissolve or otherwise remove the surroundingmicrofluidic network structure. In one particular illustrative example,a microfluidic network structure produced according to the invention andcomposed of PDMS can be filled with an epoxy prepolymer, so that theepoxy prepolymer essentially completely fills the microfluidic channelstructure of the microfluidic network. The epoxy prepolymer can then becured, for example by exposure to ultraviolet light through thesurrounding PDMS microfluidic channel structure, in order to cure theepoxy prepolymer and form a solid microstructure within the channels.The surrounding PDMS microfluidic network can then be dissolved, forexample with tetrabutylammonium fluoride (1.0 M in tetrahydrofuran)leaving behind a free-standing microstructure, comprised of epoxypolymer, having a three-dimensional structure corresponding to thethree-dimensional network of channels in the PDMS microfluidic channelstructure.

In another illustrative application for certain microfluidic channelstructures provided by the invention, the microfluidic channel structureis used as a three-dimensional microfluidic applicator or “stamp” forforming a pattern on a material surface corresponding to a pattern ofchannels disposed in one level of the microfluidic network structure.The “stamping surface” of such structures includes disposed therein aseries of channels forming indentations, which channels can delivermaterial to a substrate surface in contact with the “stamping surface”in order to form a pattern thereon corresponding to the pattern ofchannels in the stamping surface. Examples of structures discussedpreviously having “stamping surfaces” are microfluidic channel structure560 illustrated in FIG. 7 having a stamping surface 554, andmicrofluidic channel structure 760 illustrated in FIG. 9 b having astamping surface 738.

The method for patterning a material surface with a microfluidic networkstructure provided according to the invention comprises contacting astamping surface of the microfluidic network structure with a materialsurface to be stamped, and, while maintaining the stamping surface incontact with the material surface being stamped, at least partiallyfiling one or more flow paths of the microfluidic channel structure witha fluid so that at least a portion of the fluid contacts the materialsurface. Subsequently, if desired, the stamping surface can be removedfrom the material surface, yielding a pattern on the material surface,according to the pattern of channels disposed within the stampingsurface, formed by contact of the material surface with the fluid.

One example of such a stamped pattern is illustrated in FIG. 11. Themicrofluidic stamp utilized for forming the pattern in FIG. 11 waspreviously illustrated in FIG. 1 a. In forming the pattern in FIG. 11,microfluidic network 100 (FIG. 1 a) is formed so that lower surface 134is configured as a stamping surface, with the channels disposed thereincomprising indentations within the surface exposed to the externalenvironment. For embodiments wherein the microfluidic network structuresare utilized as stamps/applicators, it is especially preferred that themicrofluidic network structures be formed of an elastomeric material, sothat the stamping surface of the stamp is able to make a fluid-tightconformal seal with a wide variety of shapes and textures of materialsurfaces.

The microfluidic stamps provided according to the invention can beutilized to form patterns on material surfaces comprised of an extremelywide variety of materials, as would be apparent to those of ordinaryskill in the art. The structures provided according to the invention,when used as stamps, can be utilized, for example: to form patternedself-assembled monolayers (SAMs) on material surfaces; to form patternsof inorganic materials on surfaces; to form patterns of organic and/orbiological materials on surfaces; to form patterns on surfaces viacontacting the surfaces with a material that chemically reacts withand/or degrades/etches the material surface; etc. Essentially anymaterial able to be printed via conventional microcontact printingtechniques can be patterned onto a surface using the inventivemicrofluidic stamping structures provided by the invention. A variety ofsuch materials and applications is described in detail in U.S. Pat. Nos.5,512,131; 5,620,850; 5,776,748; 5,900,160; 5,951,881; and 5,976,826,each of which is incorporated herein by reference.

The microfluidic stamping structures provided according to the inventionhave several advantages over traditional two-dimensional microfluidicstamps. For example, the microfluidic stamping structures providedaccording to the invention have the ability to simultaneously form aplurality of patterns onto a material surface, each of which patterns iscomprised of a different material or “ink”. In general, the number ofdifferent patterns and materials which can be patterned onto a materialsurface simultaneously by the stamps provided according to the inventionis equal to the number of independent, non-fluidically interconnectedfluid flow paths disposed within the microfluidic stamping structure.

In order to form multiple patterns with different “inks” utilizingtraditional two-dimensional microcontact printing stamps, individualstamps each having a separate pattern thereon must be utilized, witheach stamp being inked with a different fluid, and with each patternbeing carefully overlaid upon the previous pattern and aligned thereto.By utilizing the three-dimensional microfluidic channel structuresprovided according to the invention, the inventive stamps are able toform, simultaneously, essentially any desired number of arbitrarilycomplex patterns on a material surface using a single stamp in a singlestamping step.

For example, referring again to FIG. 11, the microfluidic channel systemof FIG. 1 a having a stamping surface 134 is able to simultaneously forman overall pattern on material surface 900 corresponding to sevendiscrete subpatterns (A-G), each subpattern corresponding to channelsdisposed within stamping surface 134 of one of the seven fluid flowpaths (102, 104, 106, 108, 110, 112, 114) of the microfluidic channelsystem shown in FIG. 1 a. As illustrated, each of subpatterns A-Gincludes discrete pattern features (902, 904, 906, 908, 910, 912, 914,916, 918, 920, 922, and 924) which are non-continuous, and which arenon-intersecting with each other. In general, the microfluidic stampsprovided according to the invention are capable of forming patternscomprised of discrete regions, wherein the discrete regions arenon-continuous with each other, and wherein discrete regionscorresponding to and formed by channels within the stamping surface ofthe structure corresponding to two different non-fluidicallyinterconnected fluid flow paths are non-intersecting with each other.

In the illustrated pattern shown in FIG. 11, it is possible to patternup to seven different materials (“inks”) onto material surface 900simultaneously using microfluidic stamp 100 by filling each of theseparate flow paths of the microfluidic network with a different fluidafter contacting stamping surface 134 with material surface 900. Forexample, patterned regions labeled “A” in FIG. 11 can comprise a firstpatterned material, regions labeled “B” can comprise a second patternedmaterial, regions labeled “C” can comprise a third patterned material,regions labeled “D” can comprise a fourth patterned material, regionslabeled “E” can comprise a fifth patterned material, regions labeled “F”can comprise a sixth patterned material, and regions labeled “G” cancomprise a seventh patterned material. The overall pattern that resultson material surface 900 corresponds to each of the seven individualsubpatterns (A-G) formed by contact of material surface 900 with theparticular fluids contained within each of the individual flow pathsforming subpatterns A-G.

In some embodiments, regions of stamping surfaces disposed betweenchannel indentations that make conformal contact with the materialsurface being stamped can also, if desired, be coated with anothermaterial or, “ink”. In such embodiments, in addition to forming patternscorresponding to the channel structures in the stamping surface asdescribed above, the regions surrounding, contiguous with, andseparating the patterns formed by the channel structures (“printingregions”) can also contain a deposited material, carried by the printingregions, which material is printed on the material surface uponconformal contact of the “printing regions” of the stamping surface withthe material surface. The above technique enables an operator toessentially simultaneously perform a conventional microcontact printingstep and a step of depositing material in a predetermined pattern on thematerial surface via the channels disposed in the stamping surface ofthe microfluidic stamp.

Because it is possible to create arbitrarily complex patterns comprisinga large number of patterned regions containing different patternedmaterials, the stamps provided according to the invention potentiallyhave an extremely wide range of use for a wide variety of applications.For example, in one preferred application, the inventive stamps can beutilized to pattern cells and/or proteins onto surfaces. For example,proteins can be selectively patterned onto a surface which are adhesiveto cells, non-adhesive to cells, or selectively adhesive to certaincells while non-adhesive to other cells. By forming patterns with suchproteins, complex patterns of one cell type or a variety of cell typescan be selectively patterned onto surfaces for various applications, forexample, for forming biosensors or performing drug screening tests. Withthe microfluidic stamps provided according to the invention it ispossible, in principle, to pattern a large number, for example in excessof 200 or 300, different cell types, each separated from each other andarranged in a patterned array format. Such patterning can beaccomplished, according to the invention, by, for example, selectivelypatterning proteins onto a surface adherent to particular cell typesfollowed by contact of the patterned material surface with one or morecell suspensions, or by selectively patterning a plurality of differentcell types onto a surface directly using a microfluidic stamp andfilling particular fluid flow paths within the stamp with suspensionscontaining a discrete cell type or mixture of cell types desired to bepatterned onto the surface. The ability to form patterns comprisingarrays of regions, with each region including a particular cell type ormixture of cell types, can enable the creation of material surfaces foruse in biosensors or drug screening devices having cells patternedthereon that can be easily and readily identified by their spatiallocations on the surface.

Proteins can also be deposited, using the inventive microfluidic stamps,that tend to prevent or inhibit cell adhesion to a material surface.Such proteins are well known to those of ordinary skill in the art andinclude for example bovine serum albumin (BSA). In addition, proteinscan be patterned according to the invention that tend to promote celladhesion to the material surface. Such proteins include, for example,fibrinogen, collagen, laminin, integrins, antibodies, antigens, cellreceptor proteins, cell receptor antagonists, and mixtures of the above.

As described above, the microfluidic stamping structures providedaccording to the invention, can be utilized to deposit a patterned layerof cells on a material surface. Cells which can be patterned on materialsurface comprise essentially the entire range of biological cellsincluding, but not limited to, bacterial cells, algae, ameba, fungalcells, cells from multi-cellular plants, and cells from multi-cellularanimals. In some preferred embodiments, the cells comprise animal cells,and in some such embodiments comprise mammalian cells, such as humancells.

In one preferred embodiment, the mammalian cells comprise anchoragedependent cells, which can attach and spread onto material surfaces. Inone preferred embodiment, the microfluidic network stamping stampprovided according to the invention is placed with its stamping surfacein conformal contact with the material surface to be patterned with aplurality of cells, and, after filling one or more fluid flow paths ofthe microfluidic stamp with one or more suspensions of cells and beforeremoving the stamp from the material surface, the cells are allowed toincubate within the channel structure of the microfluidic stamp for aperiod of time sufficient to allow the cells to attach and spread ontothe material surface. In such an embodiment, the shape or pattern ofchannels can be specifically designed to have a predeterminedarchitecture or pattern selected to simulate a desired tissuemicro-architecture in order to study the relationship between cell shapeand/or position and cell function.

In other embodiments, two or more different cell types can be patternedonto a material surface, as described above, and, subsequent to removingthe microfluidic stamp, can be allowed to grow upon the surface andspread such that cells of the two or more different cells types spreadtogether and come into contact on the surface after a period of time haselapsed. Such a patterning and incubation method can be useful as partof an in vitro assay, which is able to determine and/or studyinteractions between different cell types. For example, such method canform part of an in vitro assay able to determine an angiogenic potentialof a particular type of tumor cell. In one particular applicationcontemplated, two different cell types comprising capillary endothelialcells and tumor cells are patterned onto a material surface and allowedto grow and spread upon the surface after patterning, as describedabove, in order to simulate and study angiogenesis during tumorformation. In vivo, tumor cells tend to attract and direct the growth ofcapillary endothelial cells to form new blood vessels to supplynutrients and oxygen for tumor growth. By forming a defined pattern ofcapillary endothelial cells and tumor cells utilizing the microfluidicstamps provided according to the invention, it can be possible to enableassays able to study the differential and competitive attraction ofcapillary endothelial cells to different tumor cell lines. Thistechnique, enabled by the present invention, can lead to the developmentof a simple, standardized, and quantitative in vitro assay for comparingthe angiogenic potential of different tumor cells.

In addition, as discussed above, the present microfluidic network stampsenable two or more different cell types to be patterned onto a materialsurface in a wide variety of patterns of arbitrary complexity and in apredetermined arrangement, which arrangement can be selected to simulatea distinct micro-architecture defined by the topological relationshipbetween the different cell types patterned on the surface. The abilityto pattern and selectively deposit different cell types in well-definedpatterned structures, enabled by the present invention, can enableassays designed to study the functional significance of tissuearchitecture at the resolution of individual cells, and can enableassays designed to study the molecular interactions between differentcell types that underlie processes such as embryonic morphogenesis,formation of the blood-brain barrier, and tumor angiogenesis.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples below. Thefollowing examples are intended to illustrate the benefits of thepresent invention, but do not exemplify the full scope of the invention.

EXAMPLE 1 Fabrication of a Mold Master by Multi-Level Photolithography

A mold master of photoresist on silicon having two levels of features inpositive, high relief (i.e., protruding above the surface of the siliconwafer) was fabricated using the two-level photolithography techniqueoutlined in FIG. 8. Designs for the channel systems for the first andsecond levels were generated with a CAD computer program (Free-Hand 8.0,MacroMedia, San Francisco, Calif.). High resolution (3386 dpi)transparencies were made by printing with a commercial printer(Linotype, Hercules Computer Technology, Inc., Freemont, Calif.) fromthe CAD computer files. Two transparencies were produced, the firstcomprising the photomask for producing-features in the first level ofthe mold master and the second comprising photomask for producing thefeatures in the second level of the mold master.

Negative photoresist (SU8-50, Microlithography Chemical Corp., Newton,Mass.) was spin-coated (at about 5,000 rpm for 20 sec) on a siliconwafer to a depth of about 50 μm and soft-baked at about 105° C. forabout 5 min to drive off solvent from the spin-cast photoresist. Thefirst transparency was then used as a photomask and the photoresist wasexposed to UV radiation for about 45 sec (wavelength of spectral linesabout: 365 nanometers, 406 nanometers, and 436 nanometers at anintensity of about 10 mW/cm²).

Without developing the uncrosslinked photoresist, a second layer ofphotoresist was spin-cast to a depth of about 100 μm on top of the firstlayer. The second transparency comprising the second photomask wasaligned to the exposed features of the photoresist of the first layerusing a Karl Suss mask aligner and exposed to the UV radiation for about1 min. The silicon wafer containing the exposed photoresist layers wasthen hard-baked for about 5 min. at about 105° C. The second photomaskcontained the pattern corresponding to the interconnecting channels thatwould eventually link channels of the first, lower level formed by thefeatures exposed through the first photomask, and channels of the upperlevels of the replica molded structure ultimately molded with the moldmaster. As illustrated in FIG. 8, each of the photomasks also included apattern for forming alignment tracks surrounding the channel system.

Both layers of photoresist were developed at the same time to removeuncrosslinked photoresist with propylene glycol methyl ether acetate.The resulting bottom master included tall alignment features and channelfeatures comprising two-level topological features in positive relief.The surface of the bottom mold master including the topological featureswas then silanized by placing the mold master in a vacuum chamber with afew drops of tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane(United Chemical Technologies, Inc., Bristol, Pa.) for about 2 hours.Silanization of the master facilitates the removal of a PDMS replicaafter molding.

EXAMPLE 2 Fabrication of a Three-Dimensional Microfluidic NetworkIncluding a System of Channels in a “Basketweave” Configuration

In the following example, the method outlined in FIGS. 9 a and 9 b wasutilized to produce a microfluidic network structure including a channelpattern therein having a basketweave structure similar to thatillustrated in FIG. 1 a. First, a bottom master was produced asdescribed above in Example 1 having disposed thereon two-leveltopological features for forming channels within the molded replicaarranged similarly to those shown schematically in FIG. 12 a by bottommaster 1000. The second step of the process comprised formation of a topmaster including features for forming channels in the uppermost level ofthe replica molded membrane. A similar schematic arrangement of featuresfor producing the channels, and the way in which the channels of theupper mold master and lower mold master fit together to mold the overallstructure, is also illustrated in FIG. 12 a, making specific referenceto upper mold master schematic 1002.

The top mold master was made by first fabricating a two-level structurein photoresist on silicon comprising a pre-master by a method similar tothat discussed above in Example 1. The pre-master contained features innegative, low-relief (i.e., comprising indentations below the level ofthe bulk surface) so that replica molding the upper mold master with thepre-master produced features in positive, high-relief on the upper moldmaster, as shown schematically in FIG. 12 a and as shown and discussedearlier in the context of FIG. 9 a. The topological features of thepre-master corresponding to the channel system extended to a level belowthe surface of the photoresist, but did not traverse it completely;these features were all on one level. Alignment tracks (not shown inFIG. 12 a) that were shaped and positioned to form alignment tracks inthe replica molded top mold master that fit between alignment tracks onthe bottom master (not shown in FIG. 12 a) during replica molding of themicrofluidic membrane with the mold masters were fabricated in deeper,negative relief and went all the way through the photoresist to thesilicon wafer. The pre-master was then silanized as described above inExample 1. The pre-master was then covered with PDMS prepolymer (Sylgard184™ silicone elastomer with about a 1:10 ratio of curing agent toelastomeric silicone polymer) and cured at about 75° C. for about 1hour. The PDMS replica, comprising a top mold master, was then peeledfrom the pre-master, trimmed, and oxidized in a plasma cleaner (PDC-23G,Harrick, Ossining, N.Y.) for 1 min, and then was silanized by placing itin a vacuum chamber with a few drops oftridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (United ChemicalTechnologies, Inc., Bristol, Pa.) for about 8 hours.

The upper mold master was then placed facedown on top of the surface ofthe bottom mold master including topological features, with a drop ofPDMS prepolymer in between. The features of the masers were alignedquickly and without magnification by manually sliding the top masterover the prepolymer and bottom master until its tall alignment tracksslipped between the tall alignment tracks of the bottom master.Utilizing PDMS for the top master enabled visual observation of thefeatures of the masters and made alignment straightforward. A microscopewas not necessary because the alignment tracks were macroscopic. Inaddition to facilitating the alignment of the segments of the channelsystem quickly and without magnification, the alignment tracks alsobalanced the top master and prevented the registered masters fromshifting in position in response to physical disturbances or theapplication of pressure during molding.

A pressure of about 100 g/mm² (1000 kPa) was then applied to the topmaster so that prepolymer did not seep between features that were incontact, and the PDMS was heated to about 75° C. and cured in place forabout 1 hour. In addition, two flat pieces of PDMS comprising an upperand lower substrate layer were formed by casting the PDMS prepolymeragainst a flat, silanized silicon wafer and curing, as described above.To transfer the membrane, the membrane and top master were peeled off asa single unit from the bottom master; the surface of the membrane andthe flat pieces of PDMS were oxidized in an air plasma for 1 min, asdescribed above; and the oxidized surfaces were then brought togetherimmediately. The oxidized PDMS surface remains reactive for a fewminutes after plasma treatment. Reactivity of the surface can beprolonged by covering the surface, if desired, with a hydrophilic liquidsuch as water, methanol, trifluoroethanol, or mixture thereof. Aprotected surface will still seal more than 30 min after oxidation.

After contacting the membrane with the bottom PDMS slab, the top masterwas peeled off, and the top surface of the membrane was sealed to thesecond oxidized flat slab to enclose the channel system. The entirestructure was then trimmed to a convenient size. The resulting structureincluded a microfluidic network incorporating eight channels in thex-direction and eight in the y-direction, each having a width of about100 μm and a height of about 70 μm, and each alternating betweencrossing over and under channels oriented perpendicular to themselves.The entire structure had a total area in the x-y plane of about 30 mm²and contained 64 crossovers.

FIG. 12 b is a photocopy of an optical photomicrograph showing an enface phase contrast image of the structure as viewed in the negativez-axis direction. The optical micrograph illustrated in FIG. 12 b wastaken of the replica molded membrane alone prior to sealing the membranebetween the upper and lower PDMS substrate layers. The opticalphotomicrograph clearly shows the basketweave microfluidic channelstructure and the crossover points of the channels, appearing asintersections in photographed the x-y plane.

After enclosing the membrane between an upper and lower PDMS supportlayer as described above, flow paths extending in the y direction werefilled with a solution of fluorescein and flow paths extending in the xdirection were filled with a solution of Meldola's Blue Dye. FIG. 12 cis a photocopy of a photomicrograph of the microfluidic channel systemfilled as described above, with the observer viewing the system en facein the negative z-axis direction. FIG. 12 c shows, without ambiguity,which channels cross over and which cross under each other, and alsodemonstrates that the channels do not intersect, as would be evidencedby mixed colors at any point.

EXAMPLE 3 Fabrication of Microstructures by Replica Molding With aMicrofluidic Network Structure

A microfluidic membrane including a three-level channel system in abasketweave pattern was produced as described in Example 2. Themicrofluidic membrane was placed upon a flat PDMS slab so that the uppersurface of the PDMS slab and the lower surface of the membrane were inconformal contact but were not irreversibly sealed to each other. Theupper surface of the membrane was left open to the atmosphere. An epoxyprepolymer (EP-TEK, Epoxy Technology, Billerica, Mass.) was then placedat the channel openings and allowed to fill the channel structure bycapillary action. After approximately 1 hour standing at ambientpressure, the epoxy had degassed and filled the channels completely. Thefilled channels were then exposed to UV light (as described above inExample 1) for about 20 min through the PDMS. The surrounding PDMSmicrofluidic membrane was then dissolved in tetrabutylammonium fluoride(1.0 M in tetrahydrofuran). FIG. 12 d is a photocopy of a scanningelectron photomicrograph of the resulting microstructure produced by thecured epoxy polymer.

EXAMPLE 4 Fabrication of a Microfluidic Network Structure Including aCoiled Fluid Flow Path Surrounding a Straight Channel

To demonstrate the capability of stacking, registering, and sealingmembranes to each other to make structures having more than three levelsof channels, a structure was fabricated including a straight channelsurrounded by a coiled fluid flow path comprising a series ofinterconnected channels. The flow path comprising the straight channelwas separated from the channels comprising the coiled flow path by athin, about 65-100 μm, PDMS layer. Examples of microfluidic systems thatbenefit from such a configuration include heat exchange elements orcountercurrent extraction system taking advantage of the diffusion ofsmall molecules across the PDMS layer separating the straight channeland the coiled fluid flow path. Multi-layer fabrication techniques suchas the one in the current example also have utility for devices forsorting and binding particles, and for complex channel network systemsthat have specific size constraints.

The method used for producing the five-level channel system by stackingand aligning two replica molded multi-level membranes was illustratedabove in FIG. 10. Referring to FIG. 10, first, bottom master 802 wasfabricated as described above in Example 1. Upper mold masters 820 and830 were fabricated as described in Example 2. Replica molded membranes800 and 810 were fabricated of cured PDMS prepolymer, also as describedabove in Example 2. Bottom master 802 was removed from each of themembranes and flat slabs of PDMS were sealed in their place, asdescribed above in Example 2. The top masters were then peeled off andthe two membranes were aligned face-to-face on the stages ofmicromanipulators. This orientation required that the two-level membrane810 be flipped over. The membranes were brought together and aligned,and were then backed apart by about 3 to about 5 mm without disturbingthe previous alignment. The separated membranes were then oxidized in anair plasma, as described above, and then brought into conformal contact.

FIG. 13 shows a photocopy of an optical photomicrograph of the resultingchannel system as viewed en face along the negative z-axis direction.Prior to the photomicrograph being taken, the two fluid flow paths ofthe system were filled with a fluorescein solution, as described inExample 2, to aid visualization of the channel system.

EXAMPLE 5 Fabrication of a Microfluidic Stamp and Etching of a Si/SiO₂Surface and Visualization of the Etched Surface Using OpticalInterference Colors

For the present example, a three-dimensional microfluidic stamp wasproduced according to the method outlined in FIG. 7. Referring to FIG.7, two-level lower mold master 520 was prepared as previously describedin Example 1 and one-level mold master 500 was prepared also asdescribed in Example 1, except utilizing only a single layer ofphotoresist and a single photomask to produce only one level oftopological features. The top PDMS slab 510 was fabricated by placingmold master 500 in a container with surface 502 facing up, covering themold master with PDMS prepolymer, curing the PDMS prepolymer, asdescribed above in Example 2, and removing and trimming the moldedreplica to form PDMS slab 510.

PDMS membrane 550 was fabricated by sandwiching a drop of PDMSprepolymer between master 520 and a PTFE sheet. Pressure of betweenabout 10 and about 50 kPa was applied tending to force the PTFE sheetand mold master 520 together. The PDMS prepolymer was then cured, asdescribed in Example 2. After curing, PTFE sheet 540 was peeled away,leaving the membrane remaining attached to mold master 520 by van derWaals interactions.

To align and seal the PDMS slab to the PDMS membrane a micromanipulatorstage was used. The slab and membrane were mounted on themicromanipulator stage so that surface 514 was facing surface 556. Thesurfaces were brought into close proximity and aligned. After alignment,the surfaces were backed away from each other by a few millimeters usingthe micromanipulator. The entire alignment stage was then placed in aplasma cleaner (Anatech, Model SP 100 Plasma System, Springfield, Va.)and oxidized for about 40 sec in an oxygen plasma. The power level ofthe plasma cleaner was about 60 watts and the oxygen pressure was about0.2 Torr. Sealing of the two layers was accomplished by removing theassembly from the plasma cleaner and immediately bringing the twoaligned and oxidized PDMS surfaces into contact.

FIG. 14 a illustrates schematically the channel system disposed in theupper level 1010 of the microfluidic stamp and the lower level 1012 ofthe microfluidic stamp, which lower level having a lower surface 554comprising the stamping surface. Surface 554 was brought into conformalcontact with material surface 1014 of substrate 1016. FIG. 14 b is aschematic diagram illustrating the layout and interconnectivity of thethree-level channel system within microfluidic stamp 560 and theconfiguration of each of the three non-fluidically interconnected fluidflow paths 561, 563, and 565.

To create the etched pattern on surface 1014 shown in FIG. 14 c, surface554 of the microfluidic stamp was brought into conformal contact withsurface 1014 (comprising a Si/SiO₂ surface) and gentle pressure wasapplied to the stamp. Three aqueous solutions containing three differentconcentrations of hydrofluoric acid (10%, 5%, and 3% hydrofluoric acid,buffered at about pH 5 with a 6:1 ratio of NH₄F/HF) were allowed to flow(˜1 cm/sec), with each solution confined to one of the non-fluidicallyinterconnected flow paths in the structure. Each of the channels in thestructure had a cross-sectional area, measured in a plane perpendicularto the channel's longitudinal axis, of about 500 μm². Where thehydrofluoric acid solutions came into contact with the surface, theyetched away the SiO₂. The rate of etching of SiO₂ for 10% hydrofluoricacid is about 20 nm/min. The lower concentrations etched at a rateproportionally less than the most concentrated solution. Thehydrofluoric acid solutions were flowed through the channels for aperiod of about 26 min before removing the stamp from the surface andvisualizing the pattern.

The optical interference color of an SiO₂ layer is very sensitive to thethickness of the layer; a difference of about 30 nm, for example, canchange the color from, for example, light green to blue. Thus, patternsetched to different depths within surface 1014 appear as differentcolors. Referring to FIG. 14 c, patterned features 1018, correspondingto fluid flow path 561, which contained the 10% hydrofluoric acidsolution, were etched into surface 1014 to a depth of about 520 nm andappear green. Etched patterned features 1020, corresponding to fluidflow path 565, which contained the 5% hydrofluoric acid solution, wereetched into surface 1014 to a depth of about 390 nm and appear yellow.Patterned features 1022, corresponding to fluid flow path 563, whichcontained the 3% hydrofluoric acid solution, were etched into surface1014 to a depth of about 70 nm and appear brown.

EXAMPLE 6 Patterned Deposition of Proteins Onto a Surface Using aThree-Dimensional Microfluidic Stamp

A microfluidic stamp having a stamping surface with spirally arrangedchannels therein was produced by a method similar to that describedabove in Example 5. The microfluidic stamp had a microfluidic channelsystem shown schematically in FIG. 15 a. The stamp included twonon-fluidically interconnected fluid flow paths 1030 and 1032. Thechannels of fluid flow paths 1030 and 1032 are disposed in the stampingsurface of the microfluidic stamp in a nested spiral arrangement asillustrated in FIG. 15 a.

The stamping surface of the microfluidic stamp was placed in conformalcontact with a polystyrene surface of a petri dish. Spiral flow paths1030 was then filled with a FITC-labeled bovine serum albumin (BSA)solution having a labeled BSA concentration of 1 mg/ml in phosphatebuffer (pH 7.4). Fluid flow path 1032 was filled with a FITC-labeledfibrinogen solution containing 0.1 mg/ml labeled fibrinogen in phosphatebuffer (pH 7.4). The proteins were allowed to absorb onto thepolystyrene surface for about 45 min. The channels were then flushedthoroughly with phosphate buffer; the stamp was peeled off; and thesurfaces were observed en face with fluorescence microscopy.

FIG. 15 b is a photocopy of a photomicrograph taken of the surface ofthe petri dish as viewed utilizing fluorescence microscopy. Spiralpattern 1034 comprises a layer of deposited labeled BSA and spiralpattern 1036 comprises a layer of deposited labeled fibrinogen. Spiralpattern 1034 is brighter and more fluorescent because the concentrationof BSA used was about 10 times higher than the concentration offluorescently labeled fibrinogen.

EXAMPLE 7 Patterned Deposition of Cells Onto Surfaces Using TwoDifferent Microfluidic Systems

Cell cultures: Bovine adrenal capillary endothelial cells (BCEs) werecultured as described in J. Folkman, C. C. Haudenschild, B. R. Zetter,Proc. Natl. Acad. Sci. USA, Vol. 76, pp. 5217-5221, 1982. In brief, BCEswere grown in low glucose DMEM cell culture medium supplemented with 10%calf serum and 2 ng/ml basic fibroblast growth factor (bFGF), and keptin a 10% CO₂ atmosphere. Human bladder cancer cells (ECVs) from theECV304 cell line were cultured in DMEM supplemented with 10% fetalbovine serum (FBS) and kept in a 5% CO₂ atmosphere. Cells from both celltypes were labeled fluorescently before harvest at 37° C. in the CO₂incubator. BCEs were incubated with1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)-conjugatedacetylated low-density lipoprotein at 4 μg/ml, which is actively takenup by BCEs and stored in endosomal granula. ECV304 cells were incubatedwith 5 μM 5-chloromethylfluorescein diacetate (CMFDA), which reacts withintracellular glutathione. Before patterning, cells were washed withPBS, dissociated from the culture plates to which they were attachedduring culture with typsin/EDTA, washed with DMEM, and resuspended inthe respective culture media at a density of about 10⁶ cells/ml. Forculturing patterned cells (both BCEs and ECVs) after removal of the PDMSstamp, DMEM supplemented with 5% calf serum, 5% FBS, and 2 ng/ml bFGFwas used, and the cells were kept in a 10% C0 ₂ atmosphere.

Patterning: To form the first pattern of deposited cells, a microfluidicstamp having the channel network structure illustrated schematically inFIG. 16 a was fabricated by a method similar to that described above inExample 5. A stamping surface of the microfluidic stamp includeddisposed therein channels comprising a concentric square pattern. Themicrofluidic stamp included three non-fluidically interconnected fluidflow paths 1040, 1042, and 1044, fluid flow path 1040 in fluidcommunication with outermost concentric square pattern 1041, fluid flowpath 1042 in fluid communication with the intermediate concentric squarepattern 1043, and fluid flow path 1044 in fluid communication with theinnermost concentric square pattern 1045.

Before use, the PDMS microfluidic stamp was autoclaved at about 121° C.for about 20 min, and the walls of the channels were coated with BSA byfilling the channels with a 0 mg/ml BSA solution in pH 7.4 phosphatebuffer for about 1 hour before removing the solution and flushing withBSA-free phosphate buffer. The stamping surface was then brought intoconformal contact with the surface of a polystyrene tissue culture dish.Suspensions of cells (at a concentration of about 5×10⁶ cells/ml) wereintroduced into the three fluid flow paths and were allowed to sedimentand attach to the surface of the tissue culture dish. The cells usedwere BCEs and an ECV cell line (ECV-304). Before being deposited, theBCEs were labeled with Dil-conjugated acetylated low-densitylipoprotein, which was actively taken up by the BCEs and stored in theirendosomal granula, and the ECVs with CMFDA, which reacted with theirintracellular glutathione. The BCE cell solutions were introduced intofluid flow paths 1040 and 1044, and the ECV cell solution was introducedinto fluid flow path 1042. After introducing the cell suspension intothe fluid flow paths of the microfluidic stamp, the cells were culturedfor about 24 hours with the microfluidic stamp in place on the tissueculture dish surface, so as to form a confluent layer of cells on thesurface of the tissue culture dish. After culture, the microfluidicstamp was removed from the surface, and the surface, having cellsattached thereto, was immersed in tissue culture media, as previouslydescribed.

FIG. 16 b is a photocopy of a photomicrograph of surface of the petridish as observed by fluorescence microscopy. The deposited BCE cells areattached to the surface in the outermost concentric square pattern 1046and the innermost concentric square pattern 1048. Such cells, whenviewed with the fluorescence microscope appear red in color. The ECVcells are deposited on the surface in concentric square pattern 1050 andfluoresce green when viewed with the fluorescence microscope. FIGS. 16 cand 16 d are photocopies of photomicrographs of the patterned surface asviewed with phase-contrast microscopy, illustrating the morphology andarrangement of the cells within each of the patterns on the surface.

FIGS. 17 a and 17 b show the results of a similar cell patterningexperiment wherein two types of cells were deposited in achessboard-like pattern. The chessboard-like pattern was designed as ademonstration of the potential of the microfluidic stamping system andmethod of the invention to deposit multiple cell types in an arrayformat appropriate for a biosensor or drug screening applications. Insuch an array, the responding cells could be identified by their spatiallocation.

A microfluidic stamp having fluid flow paths shown schematically in FIG.17 a was prepared by a method similar to that described above in Example5. The microfluidic stamp included eight non-fluidically interconnectedindependent flow paths 1060, 1062, 1064, 1066, 1068, 1070, 1072, and1074. Each of the flow paths is in fluid communication with two squarechannels disposed in the stamping surface of the microfluidic stamp. Forexample, fluid flow path 1060 is in fluid communication with squarechannels 1076 and 1078 disposed within the stamping surface of themicrofluidic stamp.

A chessboard pattern of cells is shown in FIG. 17 b, which is aphotocopy of a fluorescence photomicrograph. The patterned surface wasproduced using the same procedures used for patterning the concentricsquare pattern of FIGS. 16 b-16 d. The two cell types used, BCEs andECVs, were fluorescently labeled, as described above, before beingdeposited onto the surface of a tissue culture plate. Solutions offluorescently labeled ECV cells were used to fill fluid flow paths 1060,1062, 1064, and 1066, and solutions of fluorescently labeled BCE cellswere used to fill fluid flow paths 1068, 1070, 1072, and 1074. The cellswere cultured with the stamp in place on the surface for 42 hours untila confluent layer of cells were formed on the surface of the tissueculture plate. The fluorescence photomicrograph (a photocopy of which isshown in FIG. 17 b) was taken with the PDMS microfluidic stamp still inplace on the tissue culture plate surface in order to show theoverlaying weaving channel structures. The color of each of theconfluent layers of cells as viewed by fluorescence microscopy, isindicated on the figure above each square pattern feature. The blurredred spots 1080, 1082 and the blurred green spot 1084 comprise cellslocated in the channel structure of the top level of the microfluidicstamp above the focal plane of the microscope.

After removing the microfluidic stamp from the surface of the tissueculture plate, the surface was placed in tissue culture medium, aspreviously described, and cultured, as previously described, to allowthe two cell types to grow and spread together. FIG. 17 c shows aportion of the image of FIG. 17 b illustrating a patterned featurecomprising green deposited ECV cells and red deposited BCE cells. Thetwo regions containing cells are separated by an intermediate region ofthe tissue culture plate surface (set off by dotted white lines), whichis free of cells. FIG. 17 d shows a photocopy of a fluorescencephotomicrograph taken of the identical region of the tissue cultureplate surface taken 20 hours after removal of the stamp and subsequentculture of the plate. FIGS. 17 c and 17 d are registered, and the dottedintermediate region of FIG. 17 d comprises the region in FIG. 17 c thatwas initially cell free. As can be seen, after 20 hours of culturesubsequent to removal of the microfluidic stamp, both cell types havedivided, grown, and spread together within the region that was initiallycell free. FIG. 17 e shows the same region as shown FIG. 17 d, alsoafter 20 hours of culture subsequent to removing the stamp, except asviewed with phase contrast light microscopy.

While several embodiments of the invention have been described andillustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and structures for performing thefunctions and/or obtaing the results or advantages described herein, andeach of such variations or modifications is deemed to be within thescope of the present invention. More generally, those skilled in the artwould readily appreciate that all parameters, dimensions, materials, andconfigurations (list modified as appropriate) described herein are meantto be exemplary and that actual parameters, dimensions, materials, andconfigurations will depend upon specific applications for which theteachings of the present invention are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, theinvention may be practiced otherwise than as specifically described. Thepresent invention is directed to each individual feature, system,material and/or method described herein. In addition, any combination oftwo or more such features, systems, materials and/or methods, providedthat such features, systems, materials and/or methods are not mutuallyinconsistent, is included within the scope of the present invention. Inthe claims, all transitional phrases or phrases of inclusion, such as“comprising,” “including,” “carrying,” “having,” “containing,” and thelike are to be understood to be open-ended, i.e. to mean “including butnot limited to.” Only the transitional phrases or phrases of inclusion“consisting of” and “consisting essentially of” are to be interpreted asclosed or semi-closed phrases, respectively, as set forth in MPEPsection 2111.03.

1. A microfluidic network comprising: a polymeric structure includingtherein at least a first and a second non-fluidically interconnectedfluid flow paths, with at least the first flow path comprising a seriesof interconnected channels within the polymeric structure, the series ofinterconnected channels including at least one first channel disposedwithin a first level of the structure, at least one second channeldisposed within a second level of the structure, and at least oneconnecting channel fluidically interconnecting the first channel and thesecond channel, where at least one channel within the structure has across-sectional dimension not exceeding about 500 μm, and where thestructure includes at least one channel disposed within the first levelof the structure that is non-parallel to at least one channel disposedwithin the second level of the structure.
 2. The microfluidic network asin claim 1, wherein each of the first and second flow paths comprises aseries of interconnected channels within the polymeric structure, andwherein each series of interconnected channels includes at least onefirst channel disposed within a first level of the structure, at leastone second channel disposed within a second level of the structure, andat least one connecting channel fluidically interconnecting the firstchannel and the second channel.
 3. The microfluidic network as in claim1, wherein at least one second channel of the first flow path that isdisposed within the second level of the structure is non-parallel to atleast one first channel of the first flow path that is disposed withinthe first level of the structure.
 4. The microfluidic network as inclaim 1, wherein, at least one channel of the first fluid flow pathcrosses over at least one channel of the second fluid flow path, suchthat a perpendicular projection of the channel of the first flow pathand a perpendicular projection of the channel of the second flow pathonto a surface defining at least one of the first and second level atleast partially overlap each other.
 5. The microfluidic network as inclaim 1, wherein the polymeric structure is formed of an elastomericmaterial.
 6. The microfluidic network as in claim 5, wherein theelastomeric material comprises a silicone polymer.
 7. The microfluidicnetwork as in claim 6, wherein the silicone polymer comprisespoly(dimethylsiloxane).
 8. The microfluidic network as in claim 1,wherein the structure is comprised of at least one discrete layer ofpolymeric material.
 9. The microfluidic network as in claim 8, whereinthe structure is comprised of at least two discrete layers of polymericmaterial, each layer including at least one channel therein, the layersbeing stacked upon each other.
 10. The microfluidic network as in claim9, wherein a first discrete layer of the structure includes a surfacedefining the first level of the structure and having the at least onefirst channel disposed therein and further includes at least one channeltraversing a thickness of the layer and forming the at least oneconnecting channel, and wherein a second discrete layer of the structureincludes a surface defining the second level of the structure and havingthe at least one second channel disposed therein.
 11. The microfluidicnetwork as in claim 9, wherein the structure is comprised of at leastthree discrete layers of polymeric material, a first discrete layer ofthe structure defining the first level of the structure and having theat least one first channel disposed therein, a second discrete layer ofthe structure including at least one channel traversing a thickness ofthe layer and forming the at least one connecting channel, and a thirddiscrete layer of the structure defining the second level of thestructure and having the at least one second channel disposed therein.12. The microfluidic network as in claim 9, wherein each of the at leasttwo discrete layers is in conformal contact with another of the discretelayers.
 13. The microfluidic network as in claim 9, wherein each of theat least two discrete layers is irreversibly sealed to another of thediscrete layers.
 14. The microfluidic network as in claim 8, wherein theat least one discrete layer comprises a polymeric membrane including afirst surface defining the first level of the structure and having theat least one first channel disposed therein, a second surface definingthe second level of the structure and having the at least one secondchannel disposed therein, and a polymeric region intermediate the firstsurface and the second surface, the region including the at least oneconnecting channel therethrough fluidically interconnecting the firstchannel disposed in the first surface and the second channel disposed inthe second surface of the membrane.
 15. The microfluidic network as inclaim 14, wherein at least the first surface of the polymeric membraneis in conformal contact with a surface of a substrate.
 16. Themicrofluidic network as in claim 15, wherein the first surface of thepolymeric membrane is irreversibly sealed to the surface of thesubstrate.
 17. The microfluidic network as in claim 15, wherein thesubstrate is formed from the same material forming the polymericmembrane.
 18. The microfluidic network as in claim 15, wherein thesurface of the substrate is essentially planar.
 19. The microfluidicnetwork as in claim 15, wherein the surface of the substrate is curved.20. The microfluidic network as in claim 15, wherein the first surfaceof the polymeric membrane is in conformal contact with a surface of afirst substrate and the second surface of the polymeric membrane is inconformal contact with a surface of a second substrate.
 21. Themicrofluidic network as in claim 20, wherein the first and secondsubstrates are formed of different materials.
 22. The microfluidicnetwork as in claim 20, wherein the first and second substrates areformed of the same material.
 23. The microfluidic network as in claim22, wherein the material forming the first and second substrates is thesame as the material forming the polymeric membrane.
 24. Themicrofluidic network as in claim 20, wherein the first surface of thepolymeric membrane is irreversibly sealed to the surface of the firstsubstrate.
 25. The microfluidic network as in claim 24, wherein thesecond surface of the polymeric membrane is irreversibly sealed to thesurface of the second substrate.
 26. The microfluidic network as inclaim 14, wherein the microfluidic network comprises a plurality ofdiscrete layers comprising a plurality of polymeric membranes stackedone upon another.
 27. The microfluidic network as in claim 1, wherein atleast one channel within the structure has a cross-sectional dimensionnot exceeding about 250 μm.
 28. The microfluidic network as in claim 27,wherein at least one channel within the structure has a cross-sectionaldimension not exceeding about 100 μm.
 29. The microfluidic network as inclaim 28, wherein at least one channel within the structure has across-sectional dimension not exceeding about 50 μm.
 30. Themicrofluidic network as in claim 29, wherein at least one channel withinthe structure has a cross-sectional dimension not exceeding about 20 μm.31. A microfluidic network comprising: a polymeric structure includingtherein at least a first and a second non-fluidically-interconnectedfluid flow paths, the first flow path comprising at least twonon-colinear, interconnected channels disposed within a first plane andthe second flow path comprising at least one channel disposed within asecond plane that is non-parallel with the first plane, and where atleast one channel within the structure has a cross-sectional dimensionnot exceeding about 500 μm.
 32. The microfluidic network as in claim 31,wherein the second flow path comprises at least two non-colinear,interconnected channels defining the second plane.
 33. The microfluidicnetwork as in claim 31, wherein at least one of the first and secondflow paths comprises at least a first, a second, and a thirdinterconnected channels, the first and second channels defining togethera plane intersected by the third channel.